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.

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)

Angiotensin II receptor subtypes in cultured rat renal mesangial cells

1992 ◽  
Vol 263 (3) ◽  
pp. F411-F416 ◽  
Author(s):  
P. Ernsberger ◽  
J. Zhou ◽  
T. H. Damon ◽  
J. G. Douglas
Keyword(s):  
Binding Sites ◽  
Mesangial Cells ◽  
Specific Binding ◽  
Receptor Subtypes ◽  
Ang Ii ◽  
Angiotensin Ii Receptor ◽  
Specific Binding Sites ◽  
Angiotensin Ii Receptor Subtypes ◽  
G Protein Coupled ◽  
Two Populations

The selective angiotensin (ANG II) antagonists losartan (DuP 753) and PD 123319 have been shown to bind selectively to AT1 and AT2 subtypes, respectively. To characterize ANG II receptor subtypes in mesangial cells, washed membranes were incubated with 0.1 to 0.5 nM 125I-ANG II and increasing concentrations of competitors. The inhibition of 125I-ANG II binding by losartan and PD 123319 was biphasic, and LIGAND curve-fitting analysis revealed two populations of specific binding sites. One subpopulation comprised 86% of the total and showed high affinity for ANG II and losartan, but low affinity for the AT2 antagonists PD 123319 and CGP42112A, and thus appear identical to the recently cloned AT1 subtype. The remaining 14% of the sites showed nearly 100-fold lower affinity for losartan and 10,000-fold higher affinity for PD 123319 relative to AT1 sites. However, another AT2-selective antagonist, CGP42112A, showed little affinity for these sites. Both classes of binding sites were inhibited by guanosine 5'-O-(3-thiophosphate) and pertussis toxin treatment. We propose that there are two distinct G protein-coupled ANG II receptor subtypes (AT1A and AT1B) present in renal mesangial cells.

Increased angiotensin II receptors in neuronal cultures from hypertensive rat brain

1984 ◽  
Vol 247 (5) ◽  
pp. C364-C372 ◽  
Author(s):  
M. K. Raizada ◽  
T. F. Muther ◽  
C. Sumners
Keyword(s):  
Angiotensin Ii ◽  
Rat Brain ◽  
Binding Sites ◽  
Specific Binding ◽  
Neuronal Cell ◽  
Neuronal Cultures ◽  
Scatchard Analysis ◽  
Ang Ii ◽  
Spontaneously Hypertensive ◽  
Wistar Kyoto

Binding of 125I-angiotensin II (ANG II) to neuronal cultures made from the brains of 1-day-old normotensive (Wistar-Kyoto, WKY) and spontaneously hypertensive (SH) rats was time dependent, saturable, reversible, and 90-95% specific. Neuronal cultures from SH rats bound 50-70% more 125I-ANG II compared with their WKY controls. Scatchard analysis revealed that the increase in the specific binding of ANG II to SH rat neuronal cultures was due to an increase in the number of binding sites per cell rather than change in the affinity of receptors for ANG II. Light-microscopic autoradiographic analysis showed that ANG II specific binding sites were located on neuronal cell bodies and neurites. Treatment of neuronal cultures from both strains of rats with alpha-methyl-p-tyrosine caused a 50-60% decrease in the endogenous levels of norepinephrine (NE) and dopamine (DA). This decrease was associated with increases in the specific binding of 125I-ANG II in neuronal cultures from WKY rat brain. In contrast, ANG II binding in neuronal cultures from SH rat brain failed to respond to changes in NE and DA levels. These observations suggest that ANG II specific receptors are increased and that they are not under a negative-feedback control by catecholamines in SH rat brain neuronal cultures.

Charge heterogeneity of the AT1 angiotensin II receptor subtype in the rat lung

1995 ◽  
Vol 147 (1) ◽  
pp. 153-159 ◽  
Author(s):  
M Montiel ◽  
J Quesada ◽  
E Jiménez
Keyword(s):  
Angiotensin Ii ◽  
Specific Binding ◽  
Receptor Subtype ◽  
Scatchard Analysis ◽  
Ang Ii ◽  
Rat Lung ◽  
Angiotensin Ii Receptor ◽  
Charge Heterogeneity ◽  
Major Band ◽  
Receptor Sites

Abstract In order to obtain more information on the molecular structure of the angiotensin II (Ang II) binding sites from whole rat lung membranes these were characterized by isoelectric focusing (IEF) and SDS-PAGE. Whereas a single population of Ang II receptor sites was identified (Kd=2·2± 0·3 nmol/l; Bmax=203·9± 15·8 fmol/mg protein) by Scatchard analysis, using IEF three Ang II binding isoforms were observed; a major band which migrated to isoelectric point (pI) 6·7, and two minor bands with pI values of 6·5 and 6·3 Specific binding of 125I-Ang II to rat lung membrane preparations was sensitive to Losartan, a non-peptide AT1, receptor subtype antagonist, but was unaffected by the AT2 receptor subtype antagonist CGP42112A. Immunoblotting analyses on SDS gels, using a monoclonal antibody specific to the AT1, receptor, showed two immunoreactive protein species of 45 and 48 kDa. Enzymic deglycosylation using recombinant N-glycanase did not alter the molecular weight patterns of the AT1, receptor subtype. The results of the present study demonstrated that the Ang II receptor population in the whole rat lung consists solely of the AT1, receptor subtype and that the AT2 receptor subtype is absent. In addition, the data showed the existence of charge heterogeneity of the AT1, receptor subtype, and suggest that glycosylation probably does not contribute to its charge heterogeneity. Journal of Endocrinology (1995) 147, 153–159

Embryonic lethality in Dear gene-deficient mice: new player in angiogenesis

2005 ◽  
Vol 23 (3) ◽  
pp. 257-268 ◽  
Author(s):  
Victoria L. M. Herrera ◽  
Lorenz R. B. Ponce ◽  
Pia D. Bagamasbad ◽  
Benjamin D. VanPelt ◽  
Tamara Didishvili ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Embryonic Lethality ◽  
Female Rats ◽  
Ang Ii ◽  
Sprague Dawley ◽  
Angiotensin Ii Receptor ◽  
Endothelin 1 ◽  
Receptor Isoforms ◽  
Age Range ◽  
Deficient Mice

The dual endothelin-1/angiotensin II receptor (Dear) binds endothelin-1 (ET-1) and angiotensin II (ANG II) with equal affinities in the Dahl S/JRHS rat strain. To elucidate its physiological significance within the context of multiple receptor isoforms and diverse ET-1 and ANG II functions spanning blood pressure regulation, tumor proliferation, and angiogenesis, we characterized mouse Dear and Dear-deficient mice. Unlike null mutant models of ET-1, ANG II, and all other ET-1 and ANG II receptors, Dear−/− deficiency results in impaired angiogenesis, dysregulated neuroepithelial development, and embryonic lethality by embryonic day 12.5. Interestingly, mouse Dear does not bind ANG II, similar to Dahl R/JRHS rat Dear, but binds ET-1 and vascular endothelial growth factor (VEGF) signal peptide (VEGFsp) with equal affinities, suggesting a putative novel multifunction for VEGFsp and a parsimonious mechanism for coordination of VEGF-induced and Dear-mediated pathways. Consistent with its developmental angiogenic role, Dear inhibition results in decreased tumor growth in B16-F10 melanoma cell-induced subcutaneous tumor in female Dear+/−/C57BL6BC10 mice, but not in males (age 3.5 mo), and in 127Cs radiation-induced orthotopic mammary tumors in Sprague-Dawley female rats (age range 3–6.5 mo). Altogether, the data identify Dear as a new player in angiogenesis during development downstream to, and nonredundant with, VEGF-mediated pathways, as well as a putative modulator of tumor angiogenesis acting within a gender-specific paradigm.

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.

Regulation of angiotensin II binding sites in neuronal cultures by catecholamines

1989 ◽  
Vol 257 (4) ◽  
pp. C706-C713 ◽  
Author(s):  
L. M. Myers ◽  
C. Sumners
Keyword(s):  
Angiotensin Ii ◽  
Binding Sites ◽  
Phorbol Esters ◽  
De Novo ◽  
Specific Binding ◽  
Physiological Mechanism ◽  
Neuronal Cultures ◽  
Ang Ii ◽  
Specific Binding Sites ◽  
Stimulation Of

Previous studies determined that direct activation of protein kinase C (PKC) with phorbol esters increases the number of angiotensin II (ANG II)-specific binding sites in neuronal cultures prepared from the hypothalamus and brain stem of 1-day-old rats. In the physiological situation, PKC is activated by diacylglycerol, which can be produced by multiple pathways, such as stimulation of inositol phospholipid (IP) hydrolysis, phosphatidylcholine hydrolysis, or by de novo synthesis. In the present study we have examined whether stimulation of IP hydrolysis, and presumably activation of PKC, can mimic the actions of phorbol esters on ANG II-specific binding. We have incubated neuronal cultures with agents that increase IP hydrolysis and have determined the effects on ANG II-specific binding. Incubation of neuronal cultures with norepinephrine (NE) at concentrations (greater than 5 microM) and for times (15-60 min) that cause large increases in IP hydrolysis caused increases in the number of ANG II-specific binding sites, mimicking the actions of phorbol esters. The return of IP hydrolysis to control values was associated with a return of ANG II-specific binding to control levels. The upregulatory action of NE was abolished by prazosin, demonstrating the involvement of alpha 1-adrenergic receptors. In addition, this effect was blunted by the PKC antagonist H 7, suggesting PKC involvement in the response. Thus we have determined a potential physiological mechanism by which stimulation of IP hydrolysis by NE, and possible subsequent activation of PKC, leads to upregulation of ANG II-specific binding sites in neuronal cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II-receptor stimulation of cytosolic calcium concentration in cultured renal resistance arterioles

1996 ◽  
Vol 271 (6) ◽  
pp. F1239-F1247 ◽  
Author(s):  
Z. Zhu ◽  
W. J. Arendshorst
Keyword(s):  
Smooth Muscle ◽  
Angiotensin Ii ◽  
Vascular Reactivity ◽  
Calcium Concentration ◽  
Morphological Properties ◽  
Free Calcium ◽  
Functional Coupling ◽  
Receptor Subtypes ◽  
Ang Ii ◽  
Angiotensin Ii Receptor

This study provides an initial characterization of basic morphological properties of cultures of vascular smooth muscle cells (VSMC) from rat preglomerular resistance vessels and of the functional coupling of angiotensin II (ANG II) receptors to cytosolic free calcium concentration ([Ca2+]i (fura 2 fluorescence photometry). Renal VSMC were isolated from interlobular arteries and afferent arterioles (< 50 microns) using an iron oxide sieving method and compared with rat aortic VSMC cultured under similar conditions. Quiescent monolayers maintained uniform morphology and [Ca2+]i signaling profile between passages 3 and 10. Arteriolar and aortic VSMC were spindle shaped and expressed smooth muscle-specific alpha-actin and myosin heavy chains SM-1 and SM-2. ANG II caused a rapid increase in [Ca2+]i, followed by a sustained plateau phase at 50-60% of the peak value. The initial maximum [Ca2+]i responses were dose dependent and of similar magnitude in renal arteriolar and aortic VSMC. ANG II (10(-7) M) increased [Ca2+]i from 50 to 240 nM in arteriolar and from 57 to 201 nM in aortic VSMC (P < 0.001 for both). Inhibition of ANG II effects on [Ca2+]i revealed significant signaling through distinct AT-receptor subtypes (losartan and PD-123319 sensitive) in renal arteriolar VSMC. In contrast, only losartan was effective in aortic VSMC. The AT2-receptor ligand CGP-42112 had no effect in either vessel type. Our results demonstrate that cultured arteriolar VSMC have anatomical similarities to aortic VSMC and functional differences in AT-receptor signaling in response to ANG II. This novel preparation should provide a useful approach with which to investigate cellular mechanisms concerning receptor coupling to signaling pathways involved in vascular reactivity of arteriolar VSMC in the microcirculation in general and the kidney in particular.

Characterization of angiotensin II receptor subtypes in human glomeruli and mesangial cells

1992 ◽  
Vol 262 (3) ◽  
pp. F432-F441 ◽  
Author(s):  
D. Chansel ◽  
S. Czekalski ◽  
P. Pham ◽  
R. Ardaillou
Keyword(s):  
Angiotensin Ii ◽  
Mesangial Cells ◽  
Receptor Subtypes ◽  
Ang Ii ◽  
Glomerular Mesangial Cells ◽  
Angiotensin Ii Receptor ◽  
Functional Responses ◽  
At1 Antagonists ◽  
Angiotensin Ii Receptor Subtypes ◽  
High Concentrations

This study was designed to identify the subtypes of angiotensin II (ANG II) receptors present on glomeruli and glomerular mesangial cells and establish their functional significance. Dup 753 and its metabolite EXP 3174, two nonpeptide ANG II-1 receptor (AT1) antagonists, displaced 125I-ANG II and its analogue 125I-[Sar1,Ala8]ANG II from their binding sites in rat and human glomeruli and cultured human mesangial cells, whereas CGP 42112 A and PD 123177, two ANG II-2 receptor (AT2) antagonists, exhibited little displacing activity. Dup 753 and EXP 3174 did not modify the dissociation constant (Kd) value but markedly decreased the number of sites of 125I-[Sar1,Ala8]ANG II binding. The addition of PD 123177 did not further inhibit binding when all AT1 sites were occupied by Dup 753. Binding was markedly reduced by dithiothreitol. EXP 3174 and Dup 753 inhibited the main biological functions of ANG II in mesangial cells including increases in intracellular calcium concentration, PGE2 production, and protein synthesis. PD 123177 was also active but at concentrations 1,000- to 10,000-fold greater than those of AT1 antagonists. These results indicate that 1) only AT1 receptors are present in glomeruli and glomerular mesangial cells; 2) these receptors mediate the functional responses to ANG II; 3) the nonpeptide AT1 antagonists behave as noncompetitive inhibitors; and 4) high concentrations of the nonpeptide AT2 antagonists can recognize AT1 sites.

Sign in / Sign up

Export Citation Format

Share Document