Autoradiographic distribution in rat tissues of binding sites for endothelin: a neuropeptide?

1989 ◽  
Vol 256 (4) ◽  
pp. R858-R866 ◽  
Author(s):  
C. Koseki ◽  
M. Imai ◽  
Y. Hirata ◽  
M. Yanagisawa ◽  
T. Masaki
Keyword(s):  
Binding Sites ◽  
Rat Tissues ◽  
Binding Assays ◽  
Heart Ventricle ◽  
High Affinity ◽  
Long Acting ◽  
Porcine Endothelial Cells ◽  
Vasoconstrictor Peptide ◽  
The Brain

Endothelin (ET) is a potent and long-acting vasoconstrictor peptide consisting of 21 amino acids and recently isolated from a medium of cultured porcine endothelial cells. To determine the possible sites of ET action, we have conducted autoradiography and receptor binding assays with 125I-labeled ET in rat tissues. The displaceable binding sites of the ligand were widely distributed, not only in the arteries and heart but also in various other organs, e.g., brain, kidney, lung, adrenal gland, and intestine. The systemically injected ET did not cross the blood-brain barrier, whereas the ligand, applied in vitro, was mainly located in the hypothalamic and thalamic areas, lateral ventricular region, subfornical organ, globus pallidus, and caudate putamen. Both membrane preparations from the brain stem including diencephalon and from the heart ventricle had similar, specific, and high-affinity binding sites for 125I-ET. We suggest that ET is involved in the regulation of a large variety of organ functions and may also act as a neuropeptide.

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.

Temperature-dependence of the DnaA–DNA interaction and its effect on the autoregulation of dnaA expression

2012 ◽  
Vol 449 (2) ◽  
pp. 333-341 ◽  
Author(s):  
Chiara Saggioro ◽  
Anne Olliver ◽  
Bianca Sclavi
Keyword(s):  
Temperature Dependence ◽  
Binding Sites ◽  
Dna Interaction ◽  
Bacterial Dna ◽  
Dnaa Protein ◽  
High Affinity ◽  
Key Factor

The DnaA protein is a key factor for the regulation of the timing and synchrony of initiation of bacterial DNA replication. The transcription of the dnaA gene in Escherichia coli is regulated by two promoters, dnaAP1 and dnaAP2. The region between these two promoters contains several DnaA-binding sites that have been shown to play an important role in the negative auto-regulation of dnaA expression. The results obtained in the present study using an in vitro and in vivo quantitative analysis of the effect of mutations to the high-affinity DnaA sites reveal an additional effect of positive autoregulation. We investigated the role of transcription autoregulation in the change of dnaA expression as a function of temperature. While negative auto-regulation is lost at dnaAP1, the effects of both positive and negative autoregulation are maintained at the dnaAP2 promoter upon lowering the growth temperature. These observations can be explained by the results obtained in vitro showing a difference in the temperature-dependence of DnaA–ATP binding to its high- and low-affinity sites, resulting in a decrease in DnaA–ATP oligomerization at lower temperatures. The results of the present study underline the importance of the role for autoregulation of gene expression in the cellular adaptation to different growth temperatures.

scholarly journals Neuropilin-1 Mediates Collapsin-1/Semaphorin III Inhibition of Endothelial Cell Motility

1999 ◽  
Vol 146 (1) ◽  
pp. 233-242 ◽  
Author(s):  
Hua-Quan Miao ◽  
Shay Soker ◽  
Leonard Feiner ◽  
José Luis Alonso ◽  
Jonathan A. Raper ◽  
...  
Keyword(s):  
Binding Sites ◽  
Aortic Ring ◽  
Dependent Manner ◽  
Vascular Endothelial ◽  
Binding Assays ◽  
Angiogenesis Assay ◽  
Neuropilin 1 ◽  
Endothelial Growth Factor ◽  
Neuronal Guidance

Neuropilin-1 (NRP1) is a receptor for two unrelated ligands with disparate activities, vascular endothelial growth factor-165 (VEGF165), an angiogenesis factor, and semaphorin/collapsins, mediators of neuronal guidance. To determine whether semaphorin/collapsins could interact with NRP1 in nonneuronal cells, the effects of recombinant collapsin-1 on endothelial cells (EC) were examined. Collapsin-1 inhibited the motility of porcine aortic EC (PAEC) expressing NRP1 alone; coexpressing KDR and NRP1 (PAEC/KDR/NRP1), but not parental PAEC; or PAEC expressing KDR alone. The motility of PAEC expressing NRP1 was inhibited by 65–75% and this inhibition was abrogated by anti-NRP1 antibody. In contrast, VEGF165 stimulated the motility of PAEC/KDR/NRP1. When VEGF165 and collapsin-1 were added simultaneously to PAEC/KDR/NRP1, dorsal root ganglia (DRG), and COS-7/NRP1 cells, they competed with each other in EC motility, DRG collapse, and NRP1-binding assays, respectively, suggesting that the two ligands have overlapping NRP1 binding sites. Collapsin-1 rapidly disrupted the formation of lamellipodia and induced depolymerization of F-actin in an NRP1-dependent manner. In an in vitro angiogenesis assay, collapsin-1 inhibited the capillary sprouting of EC from rat aortic ring segments. These results suggest that collapsin-1 can inhibit EC motility as well as axon motility, that these inhibitory effects on motility are mediated by NRP1, and that VEGF165 and collapsin-1 compete for NRP1-binding sites.

RAP1 is required for BAS1/BAS2- and GCN4-dependent transcription of the yeast HIS4 gene

1991 ◽  
Vol 11 (7) ◽  
pp. 3642-3651 ◽  
Author(s):  
C Devlin ◽  
K Tice-Baldwin ◽  
D Shore ◽  
K T Arndt
Keyword(s):  
Steady State ◽  
Binding Site ◽  
Binding Sites ◽  
Binding Activity ◽  
Micrococcal Nuclease ◽  
Mrna Levels ◽  
High Affinity ◽  
Steady State Levels

The major in vitro binding activity to the Saccharomyces cerevisiae HIS4 promoter is due to the RAP1 protein. In the absence of GCN4, BAS1, and BAS2, the RAP1 protein binds to the HIS4 promoter in vivo but cannot efficiently stimulate HIS4 transcription. RAP1, which binds adjacently to BAS2 on the HIS4 promoter, is required for BAS1/BAS2-dependent activation of HIS4 basal-level transcription. In addition, the RAP1-binding site overlaps with the single high-affinity HIS4 GCN4-binding site. Even though RAP1 and GCN4 bind competitively in vitro, RAP1 is required in vivo for (i) the normal steady-state levels of GCN4-dependent HIS4 transcription under nonstarvation conditions and (ii) the rapid increase in GCN4-dependent steady-state HIS4 mRNA levels following amino acid starvation. The presence of the RAP1-binding site in the HIS4 promoter causes a dramatic increase in the micrococcal nuclease sensitivity of two adjacent regions within HIS4 chromatin: one region contains the high-affinity GCN4-binding site, and the other region contains the BAS1- and BAS2-binding sites. These results suggest that RAP1 functions at HIS4 by increasing the accessibility of GCN4, BAS1, and BAS2 to their respective binding sites when these sites are present within chromatin.

scholarly journals Binding of Dipyridamole of Human Platelets and to α-Acid Glycoprotein and its Significance for the Inhibition of Adenosine Uptake

1977 ◽  
Author(s):  
K. Subbarao ◽  
B. Rucinski ◽  
A. Summers ◽  
S. Niewiarowski
Keyword(s):  
Binding Sites ◽  
Ex Vivo ◽  
Human Platelets ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Acid Glycoprotein ◽  
High Affinity ◽  
Adenosine Uptake ◽  
Adenosine Transport

The interactions of dipyridamole with α1-acid glycoprotein of plasma and with human platelets are related to inhibition of adenosine uptake by platelets. One mole of dipyridamole binds to one mole of α1-acid glycoprotein with a dissociation constant (Kd) of 1.3 μM. It was found that platelets contain both high and low affinity binding sites for the drug. The binding of dipyridamole to the high affinity sites follows a Michaelis Menten binding pattern with a Kd of 0.04 μM. Approximately 2x104 dipyridamole molecules are bound at the high affinity sites of each platelet. The lower affinity sites bind the drug with a Kd of 4 μM. In the presence of α1acid glycoprotein the binding of dipyridamole to platelets is inhibited. Correspondingly, the dipyridamole inhibition of adenosine uptake by platelets is reduced 1000-fold by α1acid glycoprotein. Binding of dipyridamole to human platelets is essential for its inhibition of adenosine uptake by platelets. Dipyridamole reduced the [14C]-ATP to [14C]-ADP ratio in the platelets. Purified α1acid glycoprotein reversed these effects of dipyridamole on adenosine metabolism of platelets in a concentration dependent manner. A correlationwas observed between the level of circulating dipyridamole in plasma and the inhibition of [14C]-adenosine uptake by platelets of PRP samples of 12 human volunteers given different amounts of dipyridamole. The in vitro and ex vivo effects of dipyridamole on the [14C]-adenosine uptake by platelets were found to be identical. Our data suggest the presence of dipyridamole binding sites in platelets that regulate adenosine transport across the cell surface.

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.

Human macrophage maturation in vitro: Expression of functional transferrin binding sites of high affinity

1988 ◽  
Vol 57 (2) ◽  
pp. 77-83 ◽  
Author(s):  
R. Andreesen ◽  
R. G. Sephton ◽  
S. Gadd ◽  
R. C. Atkins ◽  
S. Abrew
Keyword(s):  
Binding Sites ◽  
Human Macrophage ◽  
In Vitro Expression ◽  
High Affinity ◽  
Maturation In Vitro

Bradykinin receptors localized by quantitative autoradiography in kidney, ureter, and bladder

1989 ◽  
Vol 256 (5) ◽  
pp. F909-F915 ◽  
Author(s):  
D. C. Manning ◽  
S. H. Snyder
Keyword(s):  
Guinea Pig ◽  
Binding Sites ◽  
Basal Membrane ◽  
Epithelial Layer ◽  
Bradykinin Receptors ◽  
High Affinity ◽  
Collecting Tubule ◽  
Tubule Cells

We have localized high affinity [3H]bradykinin receptor binding sites by in vitro autoradiography in kidney, ureter, and bladder of the guinea pig. The peptide pharmacology of the binding sites corresponds to that of high affinity physiological bradykinin receptors previously described (Manning, D. C., R. Vavrek, J. M. Stewart, and S. H. Snyder. J. Pharmacol. Exp. Ther. 237:504-512, 1986). In the kidney, receptors are concentrated in the medulla with negligible binding in the cortex. Medullary receptors are localized to the interstitium just beneath the basal membrane of collecting tubule cells and between tubules. In the ureter and bladder, receptors are confined to the lamina propria just beneath the epithelial layer. Localizations in the kidney may relate to the diuretic and natriuretic actions of bradykinin. Ureteral and bladder receptors may be associated with a role of bradykinin in pain and inflammation.

scholarly journals Design, Synthesis and Characterization of a New Series of Fluorescent Metabotropic Glutamate Receptor Type 5 Negative Allosteric Modulators

Molecules ◽  
2020 ◽  
Vol 25 (7) ◽  
pp. 1532
Author(s):  
Víctor Fernández-Dueñas ◽  
Mingcheng Qian ◽  
Josep Argerich ◽  
Carolina Amaral ◽  
Martijn D.P. Risseeuw ◽  
...  
Keyword(s):  
Binding Sites ◽  
Metabotropic Glutamate Receptor ◽  
Allosteric Modulators ◽  
Design Synthesis ◽  
Metabotropic Glutamate ◽  
Animal Disease Models ◽  
Fluorescent Ligands ◽  
The Brain

In recent years, new drug discovery approaches based on novel pharmacological concepts have emerged. Allosteric modulators, for example, target receptors at sites other than the orthosteric binding sites and can modulate agonist-mediated activation. Interestingly, allosteric regulation may allow a fine-tuned regulation of unbalanced neurotransmitter’ systems, thus providing safe and effective treatments for a number of central nervous system diseases. The metabotropic glutamate type 5 receptor (mGlu5R) has been shown to possess a druggable allosteric binding domain. Accordingly, novel allosteric ligands are being explored in order to finely regulate glutamate neurotransmission, especially in the brain. However, before testing the activity of these new ligands in the clinic or even in animal disease models, it is common to characterize their ability to bind mGlu5Rs in vitro. Here, we have developed a new series of fluorescent ligands that, when used in a new NanoBRET-based binding assay, will facilitate screening for novel mGlu5R allosteric modulators.

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