scholarly journals Identification of the angiotensin II receptor in rat mesenteric artery

1984 ◽  
Vol 223 (3) ◽  
pp. 659-671 ◽  
Author(s):  
J McQueen ◽  
G D Murray ◽  
P F Semple
Keyword(s):  
Angiotensin Ii ◽  
Binding Sites ◽  
Radioligand Binding ◽  
Specific Binding ◽  
Divalent Cations ◽  
Target Tissue ◽  
Angiotensin Ii Receptor ◽  
High Affinity ◽  
Rat Mesentery ◽  
Rat Mesenteric Artery

Specific binding sites of high affinity and low capacity for 125I-angiotensin II have been identified in a membrane fraction derived from arterial arcades of the rat mesentery. Heterogeneity of binding sites and extensive tracer degradation necessitated the use of nonlinear regression methods for the analysis of radioligand binding data. Forward and reverse rate constants for the high affinity sites obtained by three experimental approaches were in good agreement and gave a dissociation equilibrium constant (Kd) of 19-74 pM (95% confidence interval). Affinities for a number of angiotensin-related peptides calculated from competitive binding curves were in the order 125I-angiotensin II = angiotensin II greater than angiotensin III greater than [Sar1,Ile8]angiotensin II greater than [Sar1,Gly8]angiotensin II. Angiotensin I and biochemically unrelated peptides had virtually no effect on binding of tracer angiotensin II. The divalent cations Mn2+, Mg2+ and Ca2+ stimulated 125I-angiotensin II binding at concentrations of 2-10 mM, as did Na+ at 50-100 mM. In the presence of Na+ or Li+, K+ had a biphasic effect. The chelating agents EDTA and EGTA were inhibitory, as were the thiol reagents dithiothreitol and cysteine. This study defined angiotensin II binding sites in a vascular target tissue of sufficiently high affinity to interact rapidly with plasma angiotensin II at physiological concentrations.

Autoradiographic characterization of angiotensin II receptor subtypes in rat intestine

1993 ◽  
Vol 265 (1) ◽  
pp. G21-G27 ◽  
Author(s):  
L. A. Sechi ◽  
J. P. Valentin ◽  
C. A. Griffin ◽  
M. Schambelan
Keyword(s):  
Angiotensin Ii ◽  
Radioligand Binding ◽  
Specific Binding ◽  
Autoradiographic Study ◽  
Small Population ◽  
Photographic Emulsion ◽  
Receptor Subtypes ◽  
Ang Ii ◽  
Sprague Dawley ◽  
Angiotensin Ii Receptor

Angiotensin II is known to regulate motility and ion and water absorption in the intestine. These effects are presumed to be mediated by angiotensin II (ANG II) receptors that are present in both mucosal and muscular layers throughout the intestine. To evaluate tissue density and distribution of ANG II receptor subtypes (AT1 and AT2), we performed an in situ autoradiographic study on jejunum, ileum, and colon of Sprague-Dawley rats. Tissue sections (10 microns) were incubated with 500 pM 125I-[Sar1,Ile8]ANG II, fixed with paraformaldehyde vapors, and coated with photographic emulsion. Binding specificity was verified by competition with unlabeled [Sar1]ANG II (10 microM). AT1 and AT2 receptor distribution was characterized by competition with the nonpeptide antagonists losartan (10 microM) and PD123177 (10 microM), respectively, and the density of receptors was quantified by counting the silver grains overlying the different layers of intestinal wall. Specific binding was moderately abundant in the mucosa and the muscularis of both jejunum and ileum, whereas no binding was present in the submucosa and the serosa. Losartan inhibited 86% of radioligand binding to the mucosa in both jejunum and ileum, whereas PD123177 inhibited only 10%. The combination of the two compounds inhibited 96% of specific binding. In the colon, binding was significantly more abundant in the muscularis than in the mucosa. In this segment, losartan inhibited 90% and PD123177 16% of specific binding to muscularis. The combination of these compounds reduced binding by 97%. Thus the predominant ANG II receptor in all intestinal segments is AT1, but a small population of AT2 receptors also seems to be present.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Characterization of inositol 1,4,5-trisphosphate- and inositol 1,3,4,5-tetrakisphosphate-binding sites in rat cerebellum

1991 ◽  
Vol 274 (3) ◽  
pp. 861-867 ◽  
Author(s):  
R A J Challiss ◽  
A L Willcocks ◽  
B Mulloy ◽  
B V L Potter ◽  
S R Nahorski
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Radioligand Binding ◽  
Specific Binding ◽  
Binding Activity ◽  
Inositol Polyphosphate ◽  
Homogeneous Population ◽  
Site Model ◽  
High Affinity ◽  
Pentosan Polysulphate

1. The properties of specific Ins(1,4,5)P3- and Ins(1,3,4,5)P4-binding sites have been compared in a crude ‘P2’ cerebellar membrane fraction. 2. A homogeneous population of [3H]Ins(1,4,5)P3-binding sites was present (KD 23.1 +/- 3.6 nM) at high density (Bmax. 11.9 +/- 1.8 pmol/mg of protein); whereas data obtained for [32P]Ins(1,3,4,5)P4 specific binding were best fitted to a two-site model, the high-affinity binding component (KD 2.6 +/- 0.7 nM) constituted 64.2 +/- 4.3% of the total population and was present at relatively low density (Bmax. 187 +/- 27 fmol/mg of protein). 3. The two high-affinity inositol polyphosphate-binding sites exhibited markedly different pH optima for radioligand binding, allowing the two sites to be independently investigated. At pH 8.0, [3H]Ins(1,4,5)P3 binding was maximal, whereas [32P]Ins(1,3,4,5)P4 specific binding was very low; conversely, at pH 5.0, [32P]Ins(1,3,4,5)P4 binding was maximal, whereas [3H]Ins(1,4,5)P3 binding was undetectably low. 4. Both inositol polyphosphate-binding sites exhibited marked positional and stereo-specificity. Of the analogues studied, only phosphorothioate substitution to form inositol 1,4,5-trisphosphorothioate was tolerated at the Ins(1,4,5)P3-binding site, with only a 2-3-fold loss of binding activity. Addition of a glyceroyl moiety at the 1-phosphate position or addition of further phosphate substituents at the 3- or 6-positions caused dramatic losses in displacing activity. Similarly, complete phosphorothioate substitution of Ins(1,3,4,5)P4 caused an approx. 6-fold loss of binding activity at the [32P]Ins(1,3,4,5)P4-binding site, whereas Ins(1,4,5,6)P4, Ins(1,3,4,6)P4, Ins(1,4,5)P3 and Ins(1,3,4,5,6)P5 were bound at least 100-fold weaker at this site. Therefore, only the phosphorothioate derivatives retained high affinity and selectivity for the two inositol polyphosphate-binding sites. 5. Heparin and pentosan polysulphate were potent but non-selective inhibitors at Ins(1,4,5)P3- and Ins(1,3,4,5)P4-binding sites. N-Desulphation (with or without N-reacetylation) of heparin decreased inhibitory activity at the Ins(1,4,5)P3-, but not at the Ins(1,3,4,5)P4-binding site; however, the selectivity of this effect was only about 4-fold. O- and N-desulphated N-reacetylated heparin was essentially inactive at both sites. 6. The results are discussed with respect to the separate identities of the inositol polyphosphate-binding sites.

Binding and degradation studies on angiotensin II

1984 ◽  
Vol 62 (9) ◽  
pp. 1203-1208 ◽  
Author(s):  
A. K. Grover ◽  
C. Y. Kwan ◽  
P. Kostka ◽  
S. M. Shephard ◽  
E. E. Daniel
Keyword(s):  
Angiotensin Ii ◽  
Protease Inhibitors ◽  
Degradation Product ◽  
Binding Sites ◽  
Mesenteric Artery ◽  
Specific Binding ◽  
Parent Compound ◽  
Rat Mesenteric Artery ◽  
Major Degradation Product ◽  
Membrane Concentration

125I-labelled angiotensin II (AII) and [3H]AII showed specific binding to rat mesenteric artery microsomes. The binding in either instance was inhibited by the AII analog saralasin. [3H]AII was not degraded by the microsomes but 125I-labelled AII was degraded. Autoradiography of thin layer chromatograms of 125I-labelled AII treated with microsomes showed the parent peak (Rf = 0.4–0.45) and a single major degradation product peak (Rf = 0.25–0.30), and [125I]NaI had an Rf value higher than both 125I-labelled AII and its degradation product. Chromatography of unlabelled AII or [3H]AII gave the same Rf value as 125I-labelled AII, but unlabelled AIII moved with Rf = 0.55–0.60. The formation of the degradation product was time and membrane concentration dependent. The degradation occurred at pH 6 and 7 but not at pH 8. However, specific binding of 125I-label1ed AII was also lower at pH 8. The degradation could not be completely inhibited by the use of crude particulate fractions instead of microsomes, by preparing membranes in presence of protease inhibitors, or by including protease inhibitors and sulfhydryl agents in the assay medium. However, the degradation product neither showed specific binding to the microsomes nor interfered with the specific binding of 125I-labelled AII. Furthermore, the tightly bound material eluted from the microsomes in presence of 0.05 M acetic acid at 0 °C consisted predominantly of the parent compound. The implications of these findings are discussed both in terms of validity of the binding experiments and possible relationship between the degradation and the receptor binding sites in the membrane.

Angiotensin II receptor isoforms in the rat adrenal gland: studies with the selective subtype antagonists DuP 753 and CGP42112A

1993 ◽  
Vol 11 (1) ◽  
pp. 69-75 ◽  
Author(s):  
M Montiel ◽  
S Barker ◽  
G P Vinson ◽  
E Jiménez
Keyword(s):  
Angiotensin Ii ◽  
Adrenal Gland ◽  
Binding Sites ◽  
Specific Binding ◽  
Zona Glomerulosa ◽  
Scatchard Analysis ◽  
Receptor Subtypes ◽  
Ang Ii ◽  
Angiotensin Ii Receptor ◽  
Receptor Isoforms

ABSTRACT The angiotensin II (Ang II)-binding sites in rat adrenal gland membranes were characterized using 125I-radiolabelled Ang II. While Scatchard analysis identified a single population of Ang II receptor sites, isoelectric focusing (IEF) on polyacrylamide gels revealed four peaks of specific Ang II binding which migrated to isoelectric points (pI values) 6·8, 6·7, 6·5 and 6·3. In binding assays in the presence of an excess of the Ang II receptor AT1 subtype antagonist DuP 753, a monophasic dose-dependent displacement of 125I-labelled Ang II binding by the Ang II receptor AT2 subtype antagonist CGP42112A was observed, and vice versa. In this system, reduction of disulphide bridges using 1 mmol dithiothreitol (DTT)/l markedly increased the number of binding sites in the adrenal zona glomerulosa without affecting receptor affinity. Using IEF, it was found that both DuP 753 and CGP42112A were able to reduce specific binding of each of the four peaks to some extent. However, the predominant effect of DuP 753 was to reduce the labelling of the isoform at pI 6·7 substantially, while CGP42112A significantly inhibited the specific 125I-labelled Ang II binding to the pI 6·3 isoform. When DuP 753 and CGP42112A were used together, specific binding of 125I-labelled Ang II to the isoforms of pI values 6·8, 6·7 and 6·3 was completely eliminated. These data suggest that the four peaks of specific binding found may be composed of different isoforms of both AT1 and AT2 receptor subtypes and that the Ang II receptor isoforms which migrated to pI 6·7 and pI 6·3 are predominantly composed of AT1 and AT2 receptor subtypes respectively. Interestingly, in the presence of both antagonists, 8·7 ± 0·9% of the specific binding migrating at pI 6·5 remained unaffected. This finding suggests the presence of an additional subtype, which is neither AT1 nor AT2, in the rat adrenal zona glomerulosa. In further studies, pretreatment with DTT was found to increase the specific 125I-labelled Ang II binding of all four isoforms. Moreover, DTT also produced a further specific binding component between pI 6·5 and pI 6·7 which exhibited AT2 subtype pharmacology in DTT-treated preparations. Since DTT has been reported to enhance only AT2 subtype binding this also suggests that the different isoforms may contain components related to both AT1 and AT2 receptor subtypes.

scholarly journals Binding Affinity of Candesartan, Losartan, Telmisartan and Valsartan with Angiotensin II Receptor 1 Subtype

2013 ◽  
Vol 16 (1) ◽  
pp. 10-14 ◽  
Author(s):  
Mohiuddin Ahmed Bhuiyan ◽  
Mohammad Shahriar ◽  
Takafumi Nagatomo
Keyword(s):  
Angiotensin Ii ◽  
Binding Affinity ◽  
Binding Sites ◽  
Radioligand Binding ◽  
Pharmaceutical Journal ◽  
Angiotensin Ii Receptor ◽  
Binding Studies ◽  
At1 Receptors ◽  
At1 Antagonists ◽  
Number Of Binding Sites

This study was designed to examine the interaction in the binding of selective angiotensin II receptor antagonists towards angiotensin II type 1 receptor. The AT1 antagonists used in this study were valsartan, candesartan and losartan. Wild type AT1 receptors were transiently expressed in COS-7 cells and the expressed protein was isolated. The binding affinities of agonist and these four AT1 antagonists were determined towards AT1 receptors with the help of radioligand binding studies. The binding affinity of candesartan has been found to be maximum having a pKi value of 8.61±0.21 whereas losartan showed lowest binding affinity among the antagonists (pKi=7.17±0.07). Telmisartan also showed high (pKi=8.19±0.04) and valsartan had moderate binding affinity (pKi=7.65±0.12) towards AT1 receptors. The results of the study suggested that candesartan interacts very strongly with the receptor which is consistent with the maximum number of binding sites of in the chemical structure of candesartan. On the other hand, losartan has lower number of binding sites with the amino acid residues of AT1 receptor and as a result it showed the minimum affinity towards the receptor. DOI: http://dx.doi.org/10.3329/bpj.v16i1.14484 Bangladesh Pharmaceutical Journal 16(1): 10-14, 2013

scholarly journals Adenosine Receptors of Cerebral Microvessels and Choroid Plexus

1986 ◽  
Vol 6 (4) ◽  
pp. 463-470 ◽  
Author(s):  
Rajesh N. Kalaria ◽  
Sami I. Harik
Keyword(s):  
Cerebral Cortex ◽  
Ligand Binding ◽  
Choroid Plexus ◽  
Adenosine Receptors ◽  
Binding Sites ◽  
Specific Binding ◽  
Cerebral Microvessels ◽  
High Affinity ◽  
Receptor Sites ◽  
Ligand Binding Sites

We studied, by ligand binding methods, the two adenosine receptors, A, and A2, in rat and pig cerebral microvessels and pig choroid plexus. Ligand binding to cerebral microvessels was compared with that to membranes of the cerebral cortex. [3H]Cyclohexyladenosine and [3H]l-phenylisopropyladenosine were the ligands used for A1-receptors, and [3H]5'- N-ethylcarboxamide adenosine ([3H]NECA) was used to assess A2-receptors. We report that cerebral microvessels and choroid plexus exhibit specific [3H]NECA binding, but have no appreciable A1-receptor ligand binding sites. Specific binding of [3H]NECA to cerebral microvessels, choroid plexus, and cerebral cortex was saturable and suggested the existence of two classes of A2-receptor sites: high-affinity ( Kd ∼ 250 n M) and low-affinity ( Kd ∼ 1–2 μ M) sites. The Kd and Bmax of NECA binding to cerebral microvessels and cerebral cortex were similar within each species. Our results, indicating the existence of A2-receptors in cerebral microvessels, are consistent with results of increased adenylate cyclase activity by adenosine and some of its analogues in these microvessels.

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