Effects of vasopressin and aldosterone on amiloride binding in toad bladder epithelial cells

1975 ◽  
Vol 189 (1097) ◽  
pp. 543-575 ◽  
Keyword(s):  
Epithelial Cells ◽  
Sodium Transport ◽  
Binding Sites ◽  
Specific Binding ◽  
Sodium Concentration ◽  
Isolated Cells ◽  
The Right ◽  
Transepithelial Sodium Transport

Methods have been devised to measure the binding of [ 14 C]amiloride to isolated cells from bladders of toads, Bufo marinus . This agent blocks transepithelial sodium transport across bladders by preventing sodium entry to the transporting mechanism. A saturable binding component has been found with an affinity of 5.6 x 10 7 m -1 in the presence of 1.1 mM Na + , which corresponds to the affinity of amiloride when used as a transport inhibitor at the same sodium concentration. In freshly isolated cells the capacity of the binding sites is 3.6 x 10 5 sites/cell, but this value falls to about one third in aged suspensions. When cells are treated with vasopressin (100 mU/ml) somewhat less specific binding is measured at an amiloride concentration giving 50 % occupancy. The results are consistent with the view that vasopressin moves the binding curve to the right along the concentration axis, reducing the affinity of amiloride by a factor of approximately two, while leaving the total capacity unaffected. The affinity of amiloride when used as an inhibitor of transport is also found to be reduced by a factor of two by vasopressin, and complete inhibition of transport can still be achieved. d -Aldosterone in vitro increases the number of amiloride binding sites in isolated cells by approximately 115%, and results from transport studies indicate that there is no significant change in the affinity of amiloride after d -aldosterone treatment. Inhibitors of transcription and translation (actinomycin D and cycloheximide) prevent the increase in amiloride binding caused by d -aldosterone. In vivo the effects of d -aldosterone are more complex, but it is shown that the steroid increases the transport capacity of the tissue, when this is expressed as the number of amiloride binding sites per unit mass of tissue. The results are discussed in terms of the ways in which the two hormones may alter the entry of sodium into the epithelial cells, and so in turn affect transepithelial sodium transport.

scholarly journals Binding in vitro to rat liver receptors does not correlate with activities in vivo of bovine somatotropin. Use of chemically modified derivatives as probes

1984 ◽  
Vol 224 (2) ◽  
pp. 535-540 ◽  
Author(s):  
L P Roguin ◽  
J M Delfino ◽  
N Vita ◽  
A C Paladini
Keyword(s):  
Rat Liver ◽  
Binding Sites ◽  
Specific Binding ◽  
The Body ◽  
Water Soluble ◽  
Bovine Somatotropin ◽  
Isolated Cells ◽  
Carboxylate Groups

Bovine somatotropin with an increasing number of its carboxylate groups modified by reaction with glycine methyl ester in the presence of a water-soluble carbodi-imide was tested for its activity in different bioassays. Only those derivatives which were known to be active in the body-weight-increase bioassay were able to compete with 125I-labelled bovine somatotropin for their specific binding sites in vivo. No difference was found in the rate of clearance of a poorly active derivative as compared with that of native somatotropin. In contrast, both active and inactive derivatives were found to be equally effective in displacing the tracer from its binding sites present in isolated cells and membrane preparations from rat liver. These results suggest that the liver somatogenic receptors studied in vitro are less discriminating than those detected in vivo.

Progastrin1–80stimulates growth of intestinal epithelial cells in vitro via high-affinity binding sites

2003 ◽  
Vol 284 (2) ◽  
pp. G328-G339 ◽  
Author(s):  
P. Singh ◽  
X. Lu ◽  
S. Cobb ◽  
B. T. Miller ◽  
N. Tarasova ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Binding Sites ◽  
Intestinal Epithelial Cells ◽  
Full Length ◽  
Intestinal Epithelial ◽  
Growth Effects ◽  
High Affinity ◽  
Significant Difference

Proliferation and carcinogenesis of the large intestinal epithelial cells (IEC) cells is significantly increased in transgenic mice that overexpress the precursor progastrin (PG) peptide. It is not known if the in vivo growth effects of PG on IEC cells are mediated directly or indirectly. Full-length recombinant human PG (rhPG1–80) was generated to examine possible direct effects of PG on IEC cells. Surprisingly, rhPG (0.1–1.0 nM) was more effective than the completely processed gastrin 17 (G17) peptide as a growth factor. Even though IEC cells did not express CCK1and CCK2receptors (-R), fluorescently labeled G17 and Gly-extended G17 (G-Gly) were specifically bound to the cells, suggesting the presence of binding proteins other than CCK1-R and CCK2-R on IEC cells. High-affinity ( Kd= 0.5–1.0 nM) binding sites for125I-rhPG were discovered on IEC cells that demonstrated relative binding affinity for gastrin-like peptides in the order PG ≥ COOH-terminally extended G17 ≥ G-Gly > G17 > *CCK-8 (* significant difference; P< 0.05). In conclusion, our studies demonstrate for the first time direct growth effects of the full-length precursor peptide on IEC cells in vitro that are apparently mediated by the high-affinity PG binding sites that were discovered on these cells.

Atrial natriuretic factor binding sites in the jejunum

1989 ◽  
Vol 256 (2) ◽  
pp. G436-G441 ◽  
Author(s):  
C. Bianchi ◽  
G. Thibault ◽  
A. De Lean ◽  
J. Genest ◽  
M. Cantin
Keyword(s):  
Binding Sites ◽  
Atrial Natriuretic Factor ◽  
Specific Binding ◽  
Rat Tissues ◽  
Rat Jejunum ◽  
Specific Binding Sites ◽  
Natriuretic Factor ◽  
Atrial Natriuretic

We have studied the localization and the characterization of atrial natriuretic factor (ANF) binding sites by radioautographic techniques. Quantitative in vitro radioautography with a computerized microdensitometer demonstrated the presence of high-affinity, low-capacity 125I-ANF-(99-126) binding sites (Kd, 48 pM; Bmax, 63 fmol/mg protein) mainly in the villi of 20-microns slide-mounted transverse sections of the rat jejunum. Competition curves showed 50% inhibitory concentrations of 55 and 1,560 pM for ANF-(99-126) and ANF-(103-123), respectively. In vivo electron microscope radioautography showed that 80% of the silver grains were localized on the lamina propria fibroblast-like cells, 18% on mature enterocytes, and 2% on capillaries. Bradykinin and adrenocorticotropin did not compete with ANF binding. These results demonstrate that ANF binding sites in the rat jejunum possess the pharmacological characteristics of functional ANF receptors encountered in other rat tissues, and ultrastructural radioautographs show their cellular distribution. Taken together, these results demonstrate the presence and the localization of specific binding sites for ANF in the jejunal villi of the rat small intestine.

scholarly journals Binding of disparate transcriptional activators to nucleosomal DNA is inherently cooperative.

1995 ◽  
Vol 15 (3) ◽  
pp. 1405-1421 ◽  
Author(s):  
C C Adams ◽  
J L Workman
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Binding Sites ◽  
Specific Binding ◽  
General Mechanism ◽  
Nf Kappa B ◽  
Nucleosomal Dna ◽  
Nucleosome Binding

To investigate mechanisms by which multiple transcription factors access complex promoters and enhancers within cellular chromatin, we have analyzed the binding of disparate factors to nucleosome cores. We used a purified in vitro system to analyze binding of four activator proteins, two GAL4 derivatives, USF, and NF-kappa B (KBF1), to reconstituted nucleosome cores containing different combinations of binding sites. Here we show that binding of any two or all three of these factors to nucleosomal DNA is inherently cooperative. Thus, the binuclear Zn clusters of GAL4, the helix-loop-helix/basic domains of USF, and the rel domain of NF-kappa B all participated in cooperative nucleosome binding, illustrating that this effect is not restricted to a particular DNA-binding domain. Simultaneous binding by two factors increased the affinity of individual factors for nucleosomal DNA by up to 2 orders of magnitude. Importantly, cooperative binding resulted in efficient nucleosome binding by factors (USF and NF-kappa B) which independently possess little nucleosome-binding ability. The participation of GAL4 derivatives in cooperative nucleosome binding required only DNA-binding and dimerization domains, indicating that disruption of histone-DNA contacts by factor binding was responsible for the increased affinity of additional factors. Cooperative nucleosome binding required sequence-specific binding of all transcription factors, appeared to have spatial constraints, and was independent of the orientation of the binding sites on the nucleosome. These results indicate that cooperative nucleosome binding is a general mechanism that may play a significant role in loading complex enhancer and promoter elements with multiple diverse factors in chromatin and contribute to the generation of threshold responses and transcriptional synergy by multiple activator sites in vivo.

196 EPITHELIAL–MESENCHYMAL TRANSITION IN EQUINE AMNIOTIC PROGENITOR CELLS INDUCES CHANGES OF THE CELL GLYCAN PROFILE

2014 ◽  
Vol 26 (1) ◽  
pp. 212
Author(s):  
A. Lange-Consiglio ◽  
G. Accogli ◽  
F. Cremonesi ◽  
S. Desantis
Keyword(s):  
Epithelial Cells ◽  
Progenitor Cells ◽  
Binding Sites ◽  
Epithelial Mesenchymal Transition ◽  
Lectin Binding ◽  
Mesenchymal Transition ◽  
Glycan Profile ◽  
Amniotic Epithelial Cells

Epithelial to mesenchymal transition (EMT) is the process by which epithelial cells dramatically alter their shape and motile behaviour as they differentiate into mesenchymal cells. The EMT and the reverse process, termed mesenchymal–epithelial transition, play central roles in embryogenesis. Gastrulation and neural crest formation are processes governed by EMT in amniotes. It is noteworthy that in placental mammals, the epithelial layer of amnion originates from the trophectoderm and it is continuous with the epiblast. On this basis, it is reasonable to speculate that some amniotic epithelial cells may escape the specification that accompanies gastrulation, and may retain some of the characteristics of epiblastic cells, such as pluripotency, behaving as stem cells that are able to preserve intrinsically the ability to transdifferentiate. Because it seems that malignant cells use the same mechanisms during the formation of tumours in vivo, the amniotic epithelial cells (AEC) could represent a good model to study in vitro this phenomenon that we observed to occur spontaneously in our culture conditions. The aim of this study was to characterise the glycoprotein pattern expressed in fresh or cryopreserved equine AEC, mesenchymal (AMC), and transdifferentiated cells by means of lectin histochemistry. AEC and AMC were cultured until passage (P) 3, while transdifferentiated cells at P1(EMT1) and P2 (EMT2). All cell lines were frozen for 1 month at –196°C in liquid nitrogen. The glycoanalysis was performed with a panel of twelve lectins to detect the glycans terminating with sialic acids (MAL II, SNA, PNA after sialidase digestion (K-s), K-s-DBA), galactose (PNA, RCA120, GSA I-B4,), N-acetylgalactosamine (DBA, HPA, SBA), N-acetylglucosamine (GSA II), fucose (UEA I, LTA), or with internal mannose (Con A). After freezing: 1) AEC exhibited decrease of binding sites for DBA, SBA, HPA, GSA II, and disappearance of GSA I-B4 and UEA I binders; 2) AMC displayed increase of SBA reactivity, decrease of K-s-PNA, HPA, GSA II staining, and absence of GSA I-B4 affinity; 3) EMT1 cells showed the appearance of K-s-DBA staining, the increase of K-s-PNA, RCA120, SBA, GSA I-B4, and UEA I reactivity, the decrease of MAL II, SNA, HPA, GSA II binders, and the disappearance of DBA and LTA binding sites; 4) EMT2 cells revealed the increase of K-s-PNA, GSA I-B4, UEA I affinity, the decrease of MAL II, SNA, RCA120, HPA, GSA II binders, and the lack of DBA, SBA, and LTA reactivity. In conclusion, this study demonstrates that the EMT induces changes in cell surface glycan profile of equine amniotic progenitor cells, and for the first time revealed that freezing modifies the lectin binding pattern of these cells. The observed glycan pattern modification may represent one aspect of the spontaneous complex process of EMT.

scholarly journals The behavior of transferrin iron in the rat

Blood ◽  
1981 ◽  
Vol 57 (2) ◽  
pp. 218-228 ◽  
Author(s):  
H Huebers ◽  
W Bauer ◽  
E Huebers ◽  
E Csiba ◽  
C Finch
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Ferrous Ammonium Sulfate ◽  
Iron Binding ◽  
Body Tissues ◽  
Iron Deficient ◽  
Diferric Transferrin ◽  
Iron Exchange

Abstract The behavior of rat transferrin has been investigated employing acrylamide gel electrophoresis and isoelectric focusing. In vitro trace labeling with iron chelates at 30 min was 93%-98% effective, whereas binding by simple ferric salts was reduced to 71%-76%. Complete and specific binding of 59FeSO4 by the iron binding sites of transferrin was demonstrated after in vitro or in vivo addition of ferrous ammonium sulfate in pH 2 saline up to the point of iron saturation. In vitro the radioriron transferrin complex in plasma was stable and its iron had a negligible exchange with other transferrin binding sites over several hours. The distribution of radioiron added in vitro or through absorption was shown to be random between the binding sites of slow and fast transferrin molecule. Iron distribution among body tissues was similar for mono- and diferric transferrin iron and was not affected by the site distribution of iron on the transferrin molecule. The only important aspect of transferrin iron binding was the more rapid tissue uptake of iron in the diferric form was compared to monoferric transferrin. Additional in vivo effects on internal iron exchange were produced by changes in the iron balance of the animal. In the iron loaded animal, monoferric transferrin injected into the plasma was rapidly loaded by iron from tissue and thereby converted to diferric transferrin. Injection of diferric transferrin in the iron deficient animal was associated with a rapid disappearance from circulation of the original complex and a subsequent appearance of monoferric transferrin as a result of iron returning from tissues. These observations support the concept that plasma iron behaves as a single pool except that diferric iron exchange occurs at a more rapid rate than dose monoferric iron exchange.

scholarly journals A yeast ARS-binding protein activates transcription synergistically in combination with other weak activating factors.

1990 ◽  
Vol 10 (3) ◽  
pp. 887-897 ◽  
Author(s):  
A R Buchman ◽  
R D Kornberg
Keyword(s):  
Dna Sequences ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Essential Gene ◽  
Specific Binding ◽  
Binding Protein ◽  
Point Mutations ◽  
Yeast Genes

ABFI (ARS-binding protein I) is a yeast protein that binds specific DNA sequences associated with several autonomously replicating sequences (ARSs). ABFI also binds sequences located in promoter regions of some yeast genes, including DED1, an essential gene of unknown function that is transcribed constitutively at a high level. ABFI was purified by specific binding to the DED1 upstream activating sequence (UAS) and was found to recognize related sequences at several other promoters, at an ARS (ARS1), and at a transcriptional silencer (HMR E). All ABFI-binding sites, regardless of origin, provided weak UAS function in vivo when examined in test plasmids. UAS function was abolished by point mutations that reduced ABFI binding in vitro. Analysis of the DED1 promoter showed that two ABFI-binding sites combine synergistically with an adjacent T-rich sequence to form a strong constitutive activator. The DED1 T-rich element acted synergistically with all other ABFI-binding sites and with binding sites for other multifunctional yeast activators. An examination of the properties of sequences surrounding ARS1 left open the possibility that ABFI enhances the initiation of DNA replication at ARS1 by transcriptional activation.

scholarly journals A quantitative binding model for the Apl protein, the dual purpose recombination-directionality factor and lysis-lysogeny regulator of bacteriophage 186

2020 ◽  
Vol 48 (16) ◽  
pp. 8914-8926
Author(s):  
Erin E Cutts ◽  
J Barry Egan ◽  
Ian B Dodd ◽  
Keith E Shearwin
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Attachment Site ◽  
Binding Energies ◽  
Sequence Specificity ◽  
Binding Studies ◽  
Binding Model ◽  
Specific Binding Sites

Abstract The Apl protein of bacteriophage 186 functions both as an excisionase and as a transcriptional regulator; binding to the phage attachment site (att), and also between the major early phage promoters (pR-pL). Like other recombination directionality factors (RDFs), Apl binding sites are direct repeats spaced one DNA helix turn apart. Here, we use in vitro binding studies with purified Apl and pR-pL DNA to show that Apl binds to multiple sites with high cooperativity, bends the DNA and spreads from specific binding sites into adjacent non-specific DNA; features that are shared with other RDFs. By analysing Apl's repression of pR and pL, and the effect of operator mutants in vivo with a simple mathematical model, we were able to extract estimates of binding energies for single specific and non-specific sites and for Apl cooperativity, revealing that Apl monomers bind to DNA with low sequence specificity but with strong cooperativity between immediate neighbours. This model fit was then independently validated with in vitro data. The model we employed here is a simple but powerful tool that enabled better understanding of the balance between binding affinity and cooperativity required for RDF function. A modelling approach such as this is broadly applicable to other systems.

scholarly journals Blood to Brain Sodium Transport and Interstitial Fluid Potassium Concentration during Early Focal Ischemia in the Rat

1991 ◽  
Vol 11 (3) ◽  
pp. 466-471 ◽  
Author(s):  
Gerald P. Schielke ◽  
Hylan C. Moises ◽  
A. Lorris Betz
Keyword(s):  
Sodium Transport ◽  
Interstitial Fluid ◽  
Potassium Concentration ◽  
Capillary Endothelium ◽  
Extracellular Potassium ◽  
Sprague Dawley ◽  
Ischemic Tissue ◽  
The Right

During partial ischemia, sodium and potassium ions exchange across the blood–brain barrier, resulting in a net increase in cations and brain edema. Since this exchange is likely mediated by specific transporters such as Na,K–ATPase in the capillary endothelium and because brain capillary Na,K–ATPase activity is stimulated by increased extracellular potassium in vitro, this study was designed to determine if the rate of blood to brain sodium transport is increased in ischemic tissue having an elevated interstitial fluid potassium concentration ([K]ISF) in vivo. Sprague-Dawley rats were studied between 2–3 h after occlusion of the right middle cerebral artery. To identify where cortical tissue with an elevated [K]ISF could be sampled for transport studies, the regional pattern of cerebral blood flow and [K]ISF was obtained in a group of 17 rats using hydrogen clearance and potassium-selective microelectrode techniques. We observed severely elevated [K]ISF (> 10 m M) when CBF was less than 20 ml 100 g−1 min−1 and mildly elevated levels at CBF between 20–45 ml 100 g−1 min−1. In a second group of seven rats, permeability-surface area products (PS products) for 22Na and [3H]α-aminoisobutyric acid ([3H]AIB) were determined in ischemic cortex with elevated [K]ISF and in nonischemic cortex. The PS products for AIB were similar in both tissues (2.2 ± 0.7 and 2.1 ± 0.4 μl/g/min) while the PS products for sodium was significantly increased in the ischemic tissue (1.5 ± 0.2 and 2.4 ± 1.1 μl/g/min). This study demonstrates that blood to brain sodium transport is increased in ischemic tissue at early times before the BBB is disrupted. Stimulation of the Na,K pumps in the capillary endothelium by elevated [K]ISF may mediate this effect.

scholarly journals The behavior of transferrin iron in the rat

Blood ◽  
1981 ◽  
Vol 57 (2) ◽  
pp. 218-228
Author(s):  
H Huebers ◽  
W Bauer ◽  
E Huebers ◽  
E Csiba ◽  
C Finch
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Ferrous Ammonium Sulfate ◽  
Iron Binding ◽  
Body Tissues ◽  
Iron Deficient ◽  
Diferric Transferrin ◽  
Iron Exchange

The behavior of rat transferrin has been investigated employing acrylamide gel electrophoresis and isoelectric focusing. In vitro trace labeling with iron chelates at 30 min was 93%-98% effective, whereas binding by simple ferric salts was reduced to 71%-76%. Complete and specific binding of 59FeSO4 by the iron binding sites of transferrin was demonstrated after in vitro or in vivo addition of ferrous ammonium sulfate in pH 2 saline up to the point of iron saturation. In vitro the radioriron transferrin complex in plasma was stable and its iron had a negligible exchange with other transferrin binding sites over several hours. The distribution of radioiron added in vitro or through absorption was shown to be random between the binding sites of slow and fast transferrin molecule. Iron distribution among body tissues was similar for mono- and diferric transferrin iron and was not affected by the site distribution of iron on the transferrin molecule. The only important aspect of transferrin iron binding was the more rapid tissue uptake of iron in the diferric form was compared to monoferric transferrin. Additional in vivo effects on internal iron exchange were produced by changes in the iron balance of the animal. In the iron loaded animal, monoferric transferrin injected into the plasma was rapidly loaded by iron from tissue and thereby converted to diferric transferrin. Injection of diferric transferrin in the iron deficient animal was associated with a rapid disappearance from circulation of the original complex and a subsequent appearance of monoferric transferrin as a result of iron returning from tissues. These observations support the concept that plasma iron behaves as a single pool except that diferric iron exchange occurs at a more rapid rate than dose monoferric iron exchange.

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