scholarly journals Preparation and Biological Activity of the Monoclonal Antibody against the Second Extracellular Loop of the Angiotensin II Type 1 Receptor

2016 ◽  
Vol 2016 ◽  
pp. 1-10 ◽  
Author(s):  
Mingming Wei ◽  
Chengrui Zhao ◽  
Suli Zhang ◽  
Li Wang ◽  
Huirong Liu ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Biological Activity ◽  
Angiotensin Ii ◽  
Abdominal Cavity ◽  
Beat Frequency ◽  
Neonatal Rat ◽  
Logarithmic Phase ◽  
Extracellular Loop ◽  
Mouse Ascites

The current study was to prepare a mouse-derived antibody against the angiotensin II type 1 receptor (AT1-mAb) based on monoclonal antibody technology, to provide a foundation for research on AT1-AA-positive diseases. Balb/C mice were actively immunized with the second extracellular loop of the angiotensin II type 1 receptor (AT1R-ECII). Then, mouse spleen lymphocytes were fused with myeloma cells and monoclonal hybridomas that secreted AT1-mAb were generated and cultured, after which those in logarithmic-phase were injected into the abdominal cavity of mice to retrieve the ascites. Highly purified AT1-mAb was isolated from mouse ascites after injection with 1 × 107hybridomas. A greater amount of AT1-mAb was purified from mouse ascites compared to the cell supernatant of hybridomas. AT1-mAb purified from mouse ascites constricted the thoracic aorta of mice and increased the beat frequency of neonatal rat myocardial cells via the AT1R, identical to the effects of AT1-AA extracted from patients’ sera. Murine blood pressure increased after intravenous injection of AT1-mAb via the tail vein. High purity and good biological activity of AT1-mAb can be obtained from mouse ascites after intraperitoneal injection of monoclonal hybridomas that secrete AT1-mAb. These data provide a simple tool for studying AT1-AA-positive diseases.

Internalisation of the type I angiotensin II receptor (ATI) and angiotensin II function in the rat adrenal zona glomerulosa cell.

1994 ◽  
Vol 141 (2) ◽  
pp. R5-R9 ◽  
Author(s):  
G. P. Vinson ◽  
M. M. Ho. ◽  
J.R. Puddefoot ◽  
R. Teja ◽  
S. Barker
Keyword(s):  
Monoclonal Antibody ◽  
Plasma Membrane ◽  
Angiotensin Ii ◽  
Zona Glomerulosa ◽  
Type I ◽  
Specific Monoclonal Antibody ◽  
Angiotensin Ii Receptor ◽  
Cellular Localisation ◽  
Glomerulosa Cells

ABSTRACT Little is known about the cellular localisation of the angiotensin II (AII) type 1 receptor (ATI) in the rat adrenal glomerulosa cell, but some studies have suggested that receptor internalisation and recycling may occur. Using a specific monoclonal antibody (6313/G2) to the first extracellular domain, we show here that most of the receptor is internalised in the unstimulated cell. When viable glomerulosa cells are incubated with 6313/G2, the receptor is transiently concentrated on the cell surface, and aldosterone output is stimulated. This stimulated output is enhanced by neither threshold nor maximal stimulatory concentrations of All amide, although the antibody does not inhibit All binding to the receptor. Conversely, the stimulatory actions of the antibody and those of ACTH are additive. The data suggest that recycling to the plasma membrane is constitutive, or regulated by unknown factors. Retention of the ATI receptor in the membrane is alone enough to allow sufficient G protein interaction to generate maximal stimulatory events.

Cysteinyl leukotriene-dependent [Ca2+]iresponses to angiotensin II in cardiomyocytes

2003 ◽  
Vol 284 (4) ◽  
pp. H1269-H1276 ◽  
Author(s):  
Pinggang Liu ◽  
Derek A. Misurski ◽  
Venkat Gopalakrishnan
Keyword(s):  
Angiotensin Ii ◽  
Single Cells ◽  
Receptor Activation ◽  
Neonatal Rat ◽  
Ang Ii ◽  
Rat Cardiomyocytes ◽  
Endothelin 1 ◽  
Neonatal Rat Cardiomyocytes ◽  
Cysteinyl Leukotriene

With the use of fura 2 measurements in multiple and single cells, we examined whether cysteinyl leukotrienes (CysLT) mediate angiotensin II (ANG II)-evoked increases in cytosolic free Ca2+ concentration ([Ca2+]i) in neonatal rat cardiomyocytes. ANG II-evoked CysLT release peaked at 1 min. The angiotensin type 1 (AT1) antagonist losartan, but not the AT2antagonist PD-123319, attenuated the elevations in [Ca2+]i and CysLT levels evoked by ANG II. Vasopressin and endothelin-1 increased [Ca2+]i but not CysLT levels. The 5-lipoxygenase (5-LO) inhibitor AA-861 and the CysLT1-selective antagonist MK-571 reduced the maximal [Ca2+]i responses to ANG II but not to vasopressin and endothelin-1. While MK-571 reduced the responses to leukotriene D4 (LTD4), the dual CysLT antagonist BAY-u9773 completely blocked the [Ca2+]i elevation to both LTD4and LTC4. These data confirm that ANG II-evoked increases, but not vasopressin- and endothelin-1-evoked increases, in [Ca2+]i involve generation of the 5-lipoxygenase metabolite CysLT. The inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] antagonist 2-aminoethoxydiphenyl borate attenuated the [Ca2+]i responses to ANG II and LTD4. Thus AT1 receptor activation by ANG II is linked to CysLT-mediated Ca2+ release from Ins(1,4,5)P3-sensitive intracellular stores to augment direct ANG II-evoked Ca2+ mobilization in rat cardiomyocytes.

scholarly journals Changes in angiotensin II type 1 receptor signalling pathways evoked by a monoclonal antibody raised to the N-terminus

2008 ◽  
Vol 197 (1) ◽  
pp. 25-33 ◽  
Author(s):  
Fang Xiao ◽  
John R Puddefoot ◽  
Stewart Barker ◽  
Gavin P Vinson
Keyword(s):  
Monoclonal Antibody ◽  
Angiotensin Ii ◽  
Primary Cultures ◽  
Activator Protein 1 ◽  
Ang Ii ◽  
Reporter System ◽  
Conserved Sequence ◽  
N Terminus ◽  
Protein Kinase Cα

The extracellular N-terminus of G-protein-coupled receptors may be involved in signalling events. We examined this in the angiotensin II type 1 receptor (AT1-R) using monoclonal antibody 6313/G2, raised against a conserved sequence in the N-terminal domain, and found it evokes inhibitory and stimulatory responses. In rat aortic smooth muscle cell (RASMC) primary cultures, 6313/G2 (2.5 μg/ml) inhibited both basal and angiotensin II (Ang II; 10−7 mol/l)-stimulated [H3]thymidine incorporation. Exposure to 6313/G2 gave sustained increases in phosphorylated protein kinase Cα (PKCα) but gave a decrease in phosphorylated p44/42 extracellular signal-regulated kinases (ERK1/2) sustained from 10 min to 48 h compared with untreated control RASMC. In contrast, Ang II had no effect on PKCα, and, though it is acutely stimulatory (up to 5 min), it had no sustained effect on ERK1/2 either. Using Fura-2 and microfluorimetry, 6313/G2 added alone induced a transient increase in intracellular calcium ([Ca2+]i), with a characteristic response curve different from that of Ang II itself. The antibody was without effect on an Ang II-stimulated activator protein-1 reporter system, though it reduced unstimulated reporter activity. Such discriminatory effects on intracellular signalling suggest that the AT1-R N-terminus itself might be a target for therapeutic intervention in chronic vascular disease.

scholarly journals Long Range Effect of Mutations on Specific Conformational Changes in the Extracellular Loop 2 of Angiotensin II Type 1 Receptor

2012 ◽  
Vol 288 (1) ◽  
pp. 540-551 ◽  
Author(s):  
Hamiyet Unal ◽  
Rajaganapathi Jagannathan ◽  
Anushree Bhatnagar ◽  
Kalyan Tirupula ◽  
Russell Desnoyer ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Long Range ◽  
Conformational Changes ◽  
Extracellular Loop ◽  
Range Effect ◽  
Loop 2

Effects of Angiotensin II Type 1 Receptor Antagonist and Temperature on Prolonged Cardioplegic Arrest in Neonatal Rat Myocytes

2013 ◽  
Vol 37 (8) ◽  
pp. 689-694 ◽  
Author(s):  
Gianluca Lucchese ◽  
Giulia Elisa Cambi ◽  
Fabrizio De Rita ◽  
Mauro Franzoi ◽  
Giuseppe Faggian ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Receptor Antagonist ◽  
Neonatal Rat ◽  
Cardioplegic Arrest ◽  
Rat Myocytes

Agonistic autoantibodies directed against the angiotensin II AT1 receptor in patients with preeclampsia

2003 ◽  
Vol 81 (2) ◽  
pp. 79-83 ◽  
Author(s):  
Gerd Wallukat ◽  
Dajana Neichel ◽  
Eberhard Nissen ◽  
Volker Homuth ◽  
Friedrich C Luft
Keyword(s):  
Angiotensin Ii ◽  
Neonatal Rat ◽  
Ang Ii ◽  
Extracellular Loop ◽  
Rat Cardiomyocytes ◽  
Clinical Criteria ◽  
Neonatal Rat Cardiomyocytes ◽  
Immuno Globulins ◽  
Igg3 Subclass ◽  
Agonistic Autoantibodies

We showed that sera from patients with preeclampsia contain autoantibodies directed against the angiotensin II AT1 receptor. The antibodies recognize an epitope on the second extracellular loop of the receptor and are immuno globulins of the IgG3 subclass. The antibodies accelerate the beating rate of neonatal rat cardiomyocytes. The agonistic effect can be blocked with the AT1 receptor blocker losartan and can be neutralized by a peptide corresponding to the AT1 receptor's second extracellular loop. In further studies we shown that the autoantibodies recognize a specific conformation of the AT1 receptor. Cleavage of the external disulfide bond with dithiothreitol caused an inactivation of the receptor when stimulated either with Ang II or the autoantibodies in a system of cultured neonatal rat cardiomyocytes. Long-term stimulation of the AT1 receptor with either agonists down-regulated the AT1 receptor-mediated response to a second Ang II stimulation. These observations show that the agonistic autoantibodies behave pharmacologically in a similar fashion to Ang II. We have found the autoantibodies in all women meeting the clinical criteria of preeclampsia and suggest that they may be important to the pathogenesis of the disease.Key words: angiotensin II, preeclampsia, autoantibodies, IgG subclasses, dithiotrietol, AT1 receptor.

Abstract P090: The Contractile Effect of Angiotensin II Type 1 Receptor Autoantibodies on Human Placental Vessels

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Li Song ◽  
Ronghua Zheng ◽  
Suli Zhang ◽  
Kehua Bai ◽  
Lihong Yang ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Vascular Ring ◽  
Dependent Manner ◽  
Extracellular Loop ◽  
Maximal Contraction ◽  
Concentration Dependent Manner ◽  
Placental Perfusion ◽  
Contraction Amplitude ◽  
Placental Blood

Background: Decreased placental perfusion induced by abnormal placental vascular contraction is one of the pathological basis of preeclampsia. It has been reported that the sera titers of the autoantibody against the second extracellular loop of angiotensin II type 1 receptor (AT1-AA) were negatively correlated with placental blood flow in preeclampsia. Our previous study has found that AT1-AA could induce contraction of rat thoracic aorta and coronary rings by activing angiotensin II type 1 receptor (AT1R). However, there is no direct evidence for explaining whether AT1-AA might cause vasoconstriction on human placental blood vessels. Methods: The SD rats were immunized with the synthetic peptide corresponding to the sequence of the second extracellular loop of the human AT1 receptor (AT1R-EC II ), and anti-AT1R antibody (AT1R-Ab) was extracted. The expression of AT1R on human placental vessels was determined by immunohistochemistry. The effects of AT1R-Ab on placental vessels were measured with isolated vascular ring technique. Results: (1) AT1R was highly expressed in the human placental artery, vein and vascular endothelial cells. (2) AT1R-Ab (10 mmol/L) respectively enhanced the contraction of the placental arteries and veins (29.21% ± 3.7% vs . 21.35% ± 2.8%, P >0.05), which could be completely reversed when AT1R was blocked by AT1R inhibitor. (3) AT1R-Ab (0.1, 1 and 10 mmol/L) induced placental vasoconstriction of normal human. The percentage of maximal contraction was 2.73% ± 1.11%, 4.00% ± 3.2% and 33.30% ± 5.6%, respectively. There was significantly difference between the three groups for contraction amplitude induced by different concentrations of AT1R-Ab ( P <0.01). (4) AT1R-Ab (0.1, 1 and 10 mmol/L) induced placental vasoconstriction of preeclampsia. The percentage of maximal contraction was 1.74% ± 0.3%, 5.58% ± 1.41% and 3.73% ± 2.5%, respectively. There was significantly difference between the three groups for contraction amplitude induced by different concentrations of AT1R-Ab ( P <0.01). Conclusion: AT1R-Ab could induce human placental vasoconstriction in a concentration-dependent manner, which suggested that AT1-AA might be involved in the pathogenesis of preeclampsia by directly contracted placental blood vessles.

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