Autoradiographic detection of kinin receptors in the human medulla of control, hypertensive, and diabetic donors

2002 ◽  
Vol 80 (4) ◽  
pp. 249-257 ◽  
Author(s):  
Hudson de Sousa Buck ◽  
Brice Ongali ◽  
Gaétan Thibault ◽  
Charles J Lindsey ◽  
Réjean Couture
Keyword(s):  
Receptor Binding ◽  
Binding Sites ◽  
Specific Binding ◽  
Inferior Olivary Nucleus ◽  
Binding Studies ◽  
Specific Binding Sites ◽  
Kinin Receptors ◽  
Medullary Nuclei ◽  
Inferior Olivary

Kinins have been elected to the status of central neuromediators. Their effects are mediated through the activation of two G-protein-coupled receptors, denoted B1 and B2. Functional and binding studies suggested that B1 and B2 receptors are upregulated in the medulla and spinal cord of hypertensive and diabetic rats. The aim of this study was to localize and quantify kinin receptors in post-mortem human medulla obtained from normotensive, hypertensive, and diabetic subjects, using in vitro receptor autoradiography with the radioligands [125I]HPP-HOE140 (B2 receptor) and [125I]HPP[des-Arg10]-HOE140 (B1 receptor). Data showed specific binding sites for B2 receptor (0.4–1.5 fmol/mg tissue) in 11 medullary nuclei from 4 control specimens (paratrigeminal > ambiguus > cuneate, gelatinous layer of the caudal spinal trigeminal nucleus > caudal and interpolar spinal trigeminal, external cuneate, solitary tract > hypoglossal > gracile > inferior olivary nuclei). Increased density of B2 receptor binding sites was observed in seven medullary nuclei of four hypertensive specimens (paratrigeminal > external cuneate > interpolar and caudal spinal trigeminal, gracile, inferior olivary > hypoglossal nuclei). B2 receptor binding sites were seemingly increased in the same medullary nuclei of two diabetic specimens. Specific binding sites for B1 receptor (1.05 and 1.36 fmol/mg tissue) were seen only in the inferior olivary nucleus in two out of the ten studied specimens. The present results support a putative role for kinins in the regulation of autonomic, nociceptive, and motor functions at the level of the human medulla. Evidence is also provided that B2 receptors are upregulated in medullary cardiovascular centers of subjects afflicted of cardiovascular diseases.Key words: bradykinin, hypertension, diabetes, human brain.

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

2019 ◽  
Author(s):  
Erin Cutts ◽  
J. Barry Egan ◽  
Ian Dodd ◽  
Keith Shearwin
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Attachment Site ◽  
Binding Energies ◽  
Sequence Specificity ◽  
Binding Studies ◽  
Binding Model ◽  
Specific Binding Sites

AbstractThe 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.

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.

The binding of [3H]mepyramine to histamine H1 receptors in monkey brain

1985 ◽  
Vol 63 (6) ◽  
pp. 756-759 ◽  
Author(s):  
B. Bielkiewicz ◽  
D. A. Cook
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Histamine Receptor ◽  
Detailed Examination ◽  
Mammalian Brain ◽  
Binding Studies ◽  
Peripheral Tissues ◽  
Monkey Brain ◽  
Specific Binding Sites ◽  
Histamine H1 Receptors

Several laboratories have reported ligand binding studies using radioactive histamine H1 antagonists to label the H1 receptors in mammalian brain. We have extended these studies to a detailed examination of the binding of [3H]mepyramine to monkey brain and have shown that the distribution is similar to that in man, with specific binding sites being concentrated in the frontal cortex with relatively low binding to the pons and basal ganglia. The binding shows a single saturable component with a KD of about 1 nM and a Hill plot slope close to unity. These observations are the same for all structures tested. Comparison with data from other laboratories suggests that in this species, the histamine receptor is the same as that in peripheral tissues. From Ki values for various ligands and comparison of KD estimates in other species, the receptor seems to be essentially identical to the H1 receptor in central and peripheral tissues of the guinea pig and also to that in human brain. The rat and possibly the dog have minor differences from the monkey in terms of KD values for [3H]mepyramine binding.

Studies on Corticosterone–Receptor Complexes from Mouse Placenta

1974 ◽  
Vol 52 (3) ◽  
pp. 190-195 ◽  
Author(s):  
Ming D. Wong ◽  
A. F. Burton
Keyword(s):  
Binding Sites ◽  
Time Course ◽  
Specific Binding ◽  
Receptor Site ◽  
Binding Properties ◽  
Receptor Complexes ◽  
Specific Binding Sites ◽  
Mouse Placenta ◽  
Vitro Binding

The in vitro binding of radioactive steroids to components of mouse placental nuclei and cytoplasm was investigated using Sephadex or charcoal to remove unbound steroid. Specificity was indicated in competition experiments using excess unlabelled competing steroids. Only the active glucocorticoids formed complexes that could be isolated from the nucleus. The binding properties of the cytoplasmic steroid–receptor complex were studied. From the time course of binding the complex was shown to be more stable at 0° than at 37°, and the distribution of receptors in the cytosol appeared to be homogeneous. The complex was labile to heat and to proteolytic digestion but did not appear to be affected by nucleases or sulfhydryl reagents. Kinetic analysis revealed the presence of high affinity specific binding sites with a dissociation constant of 17.5 nM and a receptor site concentration of 0.26 pmol/mg protein. The corticosterone isolated from nuclear complexes and dexamethasone from cytoplasmic complexes were identified by chromatography and by cocrystallization as the unchanged steroid in each case.

scholarly journals Saturation Analysis in PET—Analysis of Errors Due to Imperfect Reference Regions

1994 ◽  
Vol 14 (2) ◽  
pp. 358-361 ◽  
Author(s):  
J.-E. Litton ◽  
H. Hall ◽  
S. Pauli
Keyword(s):  
Receptor Binding ◽  
Specific Binding ◽  
Scatchard Analysis ◽  
Position Emission Tomography ◽  
Reference Region ◽  
Nonspecific Binding ◽  
Binding Studies

In the determination of specific binding in receptor binding techniques in vitro as well as in vivo, determination of the nonspecific binding as well as the free component is of crucial importance. If a low proportion of specific binding is included when determining the nonspecific binding, relatively large errors may be obtained. In the present study, benzodiazepine (BZ) receptor binding in the human brain was determined in vivo using position emission tomography (PET) by applying a saturation procedure using [11C]flumazenil as an example of this problem. Analysis of the errors in Bmax and KD obtained using Scatchard analysis in PET was performed using a priori information from in vitro [3H]flumazenil binding in the pons, used normally as a reference region in BZ receptor binding studies. Even if the density of BZ receptors in the reference region pons is only 2% compared to that in the frontal cortex, this small proportion of specific binding sites will result in a 10% error in the Bmax and KD values. Simulation of a number of Scatchard plots was performed at varying ratios between the nonspecific and the specific binding.

ENDOTHELIN STIMULATES TESTOSTERONE SECRETION BY RAT LEYDIG CELLS

1993 ◽  
Vol 136 (2) ◽  
pp. R1-R4 ◽  
Author(s):  
D. Conte ◽  
P. Questino ◽  
S. Fillo ◽  
M. Nordio ◽  
A. Isidori ◽  
...  
Keyword(s):  
Binding Sites ◽  
Leydig Cells ◽  
Channel Blocker ◽  
Specific Binding ◽  
Human Chorionic Gonadotrophin ◽  
Paracrine Factor ◽  
Testosterone Secretion ◽  
Specific Binding Sites ◽  
Testicular Steroidogenesis

ABSTRACT The present study was designed to evaluate the effects of endothelin (ET) on rat testicular steroidogenesis in vitro and the involvement of prostaglandins (PG) and extracellular calcium in its mechanism of action. To this purpose we examined the effects of ET-1 and ET-3 on basal testosterone secretion, the influence of ET-1 on PGE2, release, the interaction of ET-1 and ET-3 with human chorionic gonadotrophin (hCG) and the interference of indomethacin (an inhibitor of cycloxygenase) and nifedipine (a calcium-channel blocker) in purified rat Leydig cells. The data indicate that ET-1 and ET-3 stimulate basal and hCG-induced testosterone production although the effects of ET-3 were less marked. In addition, a concomitant release of PGE2, was observed after exposure to ET-1. A sinergistic interaction between ET-1 and hCG in stimulating testicular steroidogenesis was revealed. Indomethacin was ineffective in modifying ET-1 evoked testosterone output, while in the presence of nifedipine the stimulatory effect of ET-1 was completely abolished. Since it has been shown by others that ET-1 is produced by rat Sertoli cells and specific binding sites are present in Leydig cells, the results of our study indicate that such a peptide may be regarded as a new paracrine factor able to influence steroidogenesis in Leydig cells. The action of ET-1 requires the activity of voltage-operated Ca2+ channels, while PGE2 activation is not essential for its steroidogenic effect.

Binding Sites for 125I-Labelled Endothelin-1 in the Kidneys: Differential Distribution in Rat, Pig and Man Demonstrated by Using Quantitative Autoradiography

1989 ◽  
Vol 77 (2) ◽  
pp. 129-131 ◽  
Author(s):  
Anthony P. Davenport ◽  
Derek J. Nunez ◽  
Morris J. Brown
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Receptor Autoradiography ◽  
Differential Distribution ◽  
Endothelin 1 ◽  
Specific Binding Sites ◽  
Vasa Recta ◽  
Von Willebrand ◽  
Willebrand Factor

1. Quantitative receptor autoradiography in vitro has been used to determine the distribution and density of specific binding sites for 125I-labelled endothelin-1 in human, porcine and rat kidneys. Immunocytochemistry was used to visualize von Willebrand factor-positive endothelial cells in adjacent sections to those used for autoradiography. 2. High levels of specific binding were detected in the vasa recta and papilla of all three species with lower levels in the medulla and cortex. 3. A major difference between the species was observed within the glomeruli, where high levels of binding were found in the rat but no detectable binding sites in pig or man.

Binding of [3H]des-Arg9-BK to rabbit anterior mesenteric vein

1982 ◽  
Vol 60 (12) ◽  
pp. 1551-1555 ◽  
Author(s):  
J. Barabé ◽  
C. Babiuk ◽  
D. Regoli
Keyword(s):  
Binding Sites ◽  
De Novo ◽  
Specific Binding ◽  
Kinetic Studies ◽  
Biological Action ◽  
Affinity Constant ◽  
Binding Studies ◽  
Specific Binding Sites ◽  
Pathological Conditions ◽  
Wet Weight

Binding studies of [3H]des-Arg9-BK have been performed on pieces of rabbit anterior mesenteric veins. Kinetic studies have permitted us to evaluate an affinity constant of 1.04 × 10−7 M, which is not so different from the apparent affinity constant determined by bioassay (1.6 × 10−7 M). Furthermore, inhibition of the binding of [3H]des-Arg9-BK with various kinins results in an order of potency of kinins very similar to that observed in the bioassay. Taken together, these results suggest that we are dealing with binding sites which might be the same as those subserving the biological action of des-Arg9-BK (pharmacological receptors). The preincubation of tissues in Krebs' solution brings about an increase of the specific binding from 0.06 pmol/mg of wet weight at time 0 to 0.75 pmol after 24 h; cycloheximide inhibits this increase for at least 6 h. Veins taken from animals treated with LPS, which have shown an increase in sensitivity compared with veins extracted from untreated animals, have a higher number of specific binding sites for [3H]des-Arg9-BK. The results support the hypothesis that the increased response of tissues to des-Arg9-BK is due to the de novo synthesis of receptors for kinins in some experimental and pathological conditions.

Sign in / Sign up

Export Citation Format

Share Document