P3561Detection and functional characterization of angiotensin receptor type 1 autoantibodies: establishment and clinical translation

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
V Boivin ◽  
C Zechmeister ◽  
C Schuetz ◽  
R Jahns ◽  
M J Lohse ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Intracellular Signaling ◽  
High Throughput Screening ◽  
Functional Characterization ◽  
Maximum Level ◽  
Receptor Activation ◽  
Transcriptional Level ◽  
Aldosterone Production ◽  
Signaling Inhibitors ◽  
H295r Cells

Abstract Circulating AT1R autoantibodies (AT1R-aabs) directed against the ECL2 of the AT1R with agonist-like activity are supposed to play a pathophysiological role in diseases associated with vascular and renal damage, such as preeclampsia and severehypertension (HT), but they are also thought to be involved in heart failure and primary hyperaldosteronism (PHA). Methods High-throughput screening assays aiming at a reliable detection of AT1R-aabs in sera from patients with HT and PHA were established. The agonist-like activity of AT1R-aabs was assessed by changes in intracellular calcium-levels using Fura2-QBT dye; the AlphaLISA Assay was used to assess induction of ERK1/2-phosphorylation in stably transfected AT1R-HEK-cells or in adrenocortical NCI-H295R cells. Results IgG isolated from sera of n=60 patients with PHA and n=164 with HT were screened for their capacity to increase [Ca2+]i or to activate ERK1/2. Sixteen out of 60 PHA-patients increased [Ca2+]i compared to none of the HT-patients, whereas in both disease-entities we detected AT1R-aabs inducing ERK1/2-activation with a similar prevalence (PHA: 41%, HT: 42%), indicating the existence of differentially acting AT1R-aabs. PHA-patients positive for ERK1/2-activating AT1R-aabs have significantly lower serum potassium- (3,8±0,1 vs. 4,1±0,1 mmol/l, p<0,05) and renin-levels (2,7±0,5 vs. 4,5±0,7 ng/l, p<0,05) together with an increased aldosterone concentration (341±37 vs. 236±20 ng/l, p<0,01) concordant with the disease phenotype. Similarly, higher BP values are observed in AT1R-aab positive HT-patients (syst/diast: 148/85 vs. 167/93 mmHg, p<0,0001) accompanied byhigher aldosteroneserum-levels (93±7 vs. 74±3 ng/l, p<0,05). In addition, ERK1/2-activation induced by either angiotensin II or by IgG isolated from patients with PHA or HT could be differentially blocked by the use of various signaling inhibitors. In order to elucidate if stimulating AT1R-aabs could be involved in an over-secretion of aldosterone due to sustained receptor-activation, we investigatedtheir effects on NCI-H295R-cells. At the transcriptional level, AT1R-aabs were able to induce a time-dependent upregulation of the key steroidogenic enzymes involved in aldosterone biosynthesis CYP21A1-, HSD3B2-, CYP11B1-, and in particular CYP11B2-mRNA (2fold over basal), with the maximum level achieved after 8 to 12 hours. Concordant withan agonist-stimulated internalization of AT1R,AT1R-mRNA was downregulated by AT1R-aabs (up to 25% of basal) providing direct evidence of a chronic receptor-stimulation by AT1R-aabs. Conclusion Functional assays based on AT1R-activation (Ca2+ measurements & ERK1/2-phosphorylation) are able to detect AT1R-aabs in 41% or 42% of patients with HT or PHA, respectively. Moreover, our data provide evidence that AT1R-aabs stabilize a specific AT1R-conformation distinct from that induced by angiotensin II thereby triggering a different intracellular signaling pattern resulting in chronic aldosterone production. Acknowledgement/Funding BMBF grant

Angiotensin II early regulated genes in H295R human adrenocortical cells

2004 ◽  
Vol 19 (1) ◽  
pp. 106-116 ◽  
Author(s):  
Damian G. Romero ◽  
Maria Plonczynski ◽  
Gaston R. Vergara ◽  
Elise P. Gomez-Sanchez ◽  
Celso E. Gomez-Sanchez
Keyword(s):  
Gene Expression ◽  
Angiotensin Ii ◽  
Intracellular Signaling ◽  
Zona Glomerulosa ◽  
Ang Ii ◽  
Cellular Functions ◽  
Cellular Mechanisms ◽  
Mediated Effects ◽  
Aldosterone Production ◽  
H295r Cells

Evidence for the dysregulation of aldosterone synthesis in cardiovascular pathophysiology has renewed interest in the control of its production. Cellular mechanisms by which angiotensin II (ANG II) stimulates aldosterone synthesis in the adrenal zona glomerulosa are incompletely understood. To elucidate the mechanism of intracellular signaling by ANG II stimulation in the adrenal, we have studied immediate-early regulated genes in human adrenal H295R cells using cDNA microarrays. H295R cells were stimulated with ANG II for 3 h. Gene expression was analyzed by microarray technology and validated by real-time RT-PCR. Eleven genes were found to be upregulated by ANG II. These encode the proteins for ferredoxin, Nor1, Nurr1, c6orf37, CAT-1, A20, MBLL, M-Ras, RhoB, GADD45α, and a novel protein designated FLJ45273 . Maximum expression levels for all genes occurred 3–6 h after ANG II stimulation. This increase was dose dependent and preceded maximal aldosterone production. Other aldosterone secretagogues, K+and endothelin-1 (ET-1), also induced the expression of these genes with variable efficiency depending on the gene and with lower potency than ANG II. ACTH had negligible effect on gene expression except for the CAT-1 and Nurr1 genes. These ANG II-stimulated genes are involved in several cellular functions and are good candidate effectors and regulators of ANG II-mediated effects in adrenal zona glomerulosa.

scholarly journals Homocysteine promotes migration of adventitial fibroblasts via Angiotensin II type 1 receptor activation to aggravate atherosclerosis

2020 ◽  
Author(s):  
Zhibo Zhu ◽  
Xiangyu Hao ◽  
Tianxiang Li ◽  
Jianqiang Guo
Keyword(s):  
Angiotensin Ii ◽  
Intracellular Signaling ◽  
Receptor Activation ◽  
Inflammatory Reactions ◽  
Intracellular Signaling Pathway ◽  
Plaque Area ◽  
Apolipoprotein E Gene ◽  
Adventitial Fibroblasts ◽  
One Way Anova

Abstract Background: Hyperhomocysteinemia (HHcy) is an independent risk factor for atherosclerosis. However, the mechanisms of HHcy-induced arteriosclerosis are largely unknown. Objective: To clarify the effect of Hcy on adventitial fibroblasts (AFs) and its relation with angiotensin II type 1 receptor (AT1R). Method: The apolipoprotein E gene-deficient mice (Apoe−/−) were used as the murine model for atherosclerosis. HHcy was induced and treated by feeding them 1.5% methionine and telmisartan (gavage 10 mg/kg/d) for 12 weeks, respectively. The AFs and HEK293A cells transfected with the AT1R plasmid were used to investigate the interaction between Hcy and AT1R. All data were expressed as mean ± SEM. The data were analyzed by one-way ANOVA or repeated measurement ANOVA. Results: HHcy aggravated the plaque area of the aortic root and increased the expression of IL-6, MCP-1, and the macrophage marker Mac3 in the plaque and adventitia of the aorta, whereas telmisartan improved this effect. Hyperhomocysteinemia induced the occurrence of the AFs marker protein ER-TR7 in the plaque and the entire layer of the aorta, whereas telmisartan also improved this effect, indicating that hyperhomocysteinemia induced AFs migration and that AT1R mediated this process. Hcy increased the production of AFs H 2 O 2 , ROS, and IL-6 of AFs, indicating that Hcy activated the oxidative stress and inflammatory reactions, which may induce cell migration. The subsequent scratch and transwell experiments confirmed that Hcy induced the AFs migration and that telmisartan inhibited this effect. Hcy increased the expression of AFs AT1R and the phosphorylation levels of PKC and ERK1/2 in the AF and HEK293A cells transfected with the AT1R plasmid, whereas telmisartan inhibited this effect, indicating that Hcy activated AT1R intracellular signaling pathway. Conclusion: Hcy promoted adventitial inflammation, induced AFs migration, and aggravated atherosclerosis by activating AT1R.

Long-term presence of angiotensin II type 1 receptor autoantibody reduces aldosterone production by triggering Ca2+ overload in H295R cells

2017 ◽  
Vol 66 (1) ◽  
pp. 44-51 ◽  
Author(s):  
Jinghui Lei ◽  
Suli Zhang ◽  
Pengli Wang ◽  
Yang Liao ◽  
Jingwei Bian ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Aldosterone Production ◽  
H295r Cells

scholarly journals Homocysteine Promotes Migration of Adventitial Fibroblasts via Angiotensin II type 1 Receptor Activation to Aggravate Atherosclerosis

2020 ◽  
Author(s):  
Zhibo Zhu ◽  
Xiangyu Hao ◽  
Tianxiang Li ◽  
Jianqiang Guo
Keyword(s):  
Angiotensin Ii ◽  
Intracellular Signaling ◽  
Receptor Activation ◽  
Inflammatory Reactions ◽  
Intracellular Signaling Pathway ◽  
Plaque Area ◽  
Apolipoprotein E Gene ◽  
Adventitial Fibroblasts ◽  
One Way Anova

Abstract BackgroundHyperhomocysteinemia (HHcy) is an independent risk factor for atherosclerosis. However, the mechanisms of HHcy-induced arteriosclerosis are largely unknown. Objective:To clarify the effect of Hcy on adventitial fibroblasts (AFs) and its relation with angiotensin II type 1 receptor (AT1R).Method:The apolipoprotein E gene-deficient mice (Apoe−/−) were used as the murine model for atherosclerosis. HHcy was induced and treated by feeding them 1.5% methionine and telmisartan (gavage 10 mg/kg/d) for 12 weeks, respectively. The AFs and HEK293A cells transfected with the AT1R plasmid were used to investigate the interaction between Hcy and AT1R. All data were expressed as mean ± SEM. The data were analyzed by one-way ANOVA or repeated measurement ANOVA.Results:HHcy aggravated the plaque area of the aortic root and increased the expression of IL-6, MCP-1, and the macrophage marker Mac3 in the plaque and adventitia of the aorta, whereas telmisartan improved this effect. Hyperhomocysteinemia induced the occurrence of the AFs marker protein ER-TR7 in the plaque and the entire layer of the aorta, whereas telmisartan also improved this effect, indicating that hyperhomocysteinemia induced AFs migration and that AT1R mediated this process. Hcy increased the production of AFs H2O2, ROS, and IL-6 of AFs, indicating that Hcy activated the oxidative stress and inflammatory reactions, which may induce cell migration. The subsequent scratch and transwell experiments confirmed that Hcy induced the AFs migration and that telmisartan inhibited this effect. Hcy increased the expression of AFs AT1R and the phosphorylation levels of PKC and ERK1/2 in the AF and HEK293A cells transfected with the AT1R plasmid, whereas telmisartan inhibited this effect, indicating that Hcy activated AT1R intracellular signaling pathway.Conclusion:Hcy promoted adventitial inflammation, induced AFs migration, and aggravated atherosclerosis by activating AT1R.

scholarly journals Intracellular Transport of Calcium from Plasma Membrane to Mitochondria in Adrenal H295R Cells: Implication for Steroidogenesis

Endocrinology ◽  
2003 ◽  
Vol 144 (10) ◽  
pp. 4575-4585 ◽  
Author(s):  
Nathalie Lalevée ◽  
Véronique Resin ◽  
Serge Arnaudeau ◽  
Nicolas Demaurex ◽  
Michel F. Rossier
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Angiotensin Ii ◽  
Calcium Channels ◽  
Extracellular Potassium ◽  
Cell Periphery ◽  
Calyculin A ◽  
Calcium Response ◽  
Aldosterone Production ◽  
H295r Cells

Angiotensin II and extracellular potassium stimulate aldosterone production in adrenal glomerulosa cells by mobilizing the calcium messenger system. This response requires calcium influx across the plasma membrane, followed by calcium uptake into the mitochondria. It has been proposed that calcium is transported to the mitochondria via the lumen of the endoplasmic reticulum, acting as a kind of intracellular calcium pipeline. This hypothesis has been tested in the present study by measuring intramitochondrial calcium variations in H295R cells with a new fluorescent calcium probe, ratiometric pericam. Calyculin A, a protein phosphatase inhibitor, induced the formation of a large cortical layer of actin filaments, removing the peripheral endoplasmic reticulum away from the plasma membrane and thereby physically uncoupling the calcium channels from the pipeline. The mitochondrial calcium response to potassium was markedly reduced after calyculin treatment, but that of AngII was unaffected. Under the same conditions, potassium-stimulated pregnenolone and aldosterone production was significantly reduced, whereas the steroidogenic response to AngII remained unchanged. The inhibitory action of calyculin A on the responses to potassium was not mediated by a modification of the calcium channel activity and was not accompanied by a reduction of the cytosolic calcium response. It therefore appears that, in H295R cells, the organization of the actin cytoskeleton at the cell periphery influences the steroidogenic action of potassium, but not the response to angiotensin II. The response to potassium is proposed to be dependent on the endoplasmic reticulum-mediated transfer of calcium entering through plasma membrane calcium channels to the mitochondria.

scholarly journals Homocysteine promotes migration of adventitial fibroblasts via Angiotensin II type 1 receptor activation to aggravate atherosclerosis

2020 ◽  
Author(s):  
Zhibo Zhu ◽  
Xiangyu Hao ◽  
Tianxiang Li ◽  
Jianqiang Guo
Keyword(s):  
Angiotensin Ii ◽  
Intracellular Signaling ◽  
Receptor Activation ◽  
Inflammatory Reactions ◽  
Intracellular Signaling Pathway ◽  
Plaque Area ◽  
Apolipoprotein E Gene ◽  
Adventitial Fibroblasts ◽  
One Way Anova

Abstract BackgroundHyperhomocysteinemia (HHcy) is an independent risk factor for atherosclerosis. However, the mechanisms of HHcy-induced arteriosclerosis are largely unknown. Objective: To clarify the effect of Hcy on adventitial fibroblasts (AFs) and its relation with angiotensin II type 1 receptor (AT1R).Method:The apolipoprotein E gene-deficient mice (ApoE−/−) were used as the murine model for atherosclerosis. HHcy was induced and treated by feeding them 1.5% methionine and telmisartan (gavage 10 mg/kg/d) for 12 weeks, respectively. The AFs and HEK293A cells transfected with the AT1R plasmid were used to investigate the interaction between Hcy and AT1R. All data were expressed as mean ± SEM. The data were analyzed by one-way ANOVA or repeated measurement ANOVA.Results:HHcy aggravated the plaque area of the aortic root and increased the expression of IL-6, MCP-1, and the macrophage marker Mac3 in the plaque and adventitia of the aorta, whereas telmisartan improved this effect. HHcy induced the occurrence of the AFs marker protein ER-TR7 in the plaque and the entire layer of the aorta, whereas telmisartan also improved this effect, indicating that HHcy induced AFs migration and that AT1R mediated this process. Hcy increased the production of AFs H2O2, ROS, and IL-6 of AFs, indicating that Hcy activated the oxidative stress and inflammatory reactions, which may induce cell migration. The subsequent scratch and transwell experiments confirmed that Hcy induced the AFs migration and that telmisartan inhibited this effect. Hcy increased the expression of AFs AT1R and the phosphorylation levels of PKC and ERK1/2 in the AF and HEK293A cells transfected with the AT1R plasmid, whereas telmisartan inhibited this effect, indicating that Hcy activated AT1R intracellular signaling pathway.Conclusion:Hcy promoted adventitial inflammation, induced AFs migration, and aggravated atherosclerosis by activating AT1R.

scholarly journals Somatic Mutations of GNA11 and GNAQ in CTNNB1-Mutant Aldosterone-Producing Adenomas Increases Aldosterone and Aldosterone Synthase (CYP11B2)

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A72-A72
Author(s):  
Elena A B Azizan ◽  
Junhua Zhou ◽  
Claudia P Cabrera ◽  
Fabio Luiz Fernandes-Rosa ◽  
Sheerazed Boulkroun ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Double Mutant ◽  
Somatic Mutations ◽  
Wild Type ◽  
Aldosterone Secretion ◽  
Discovery Cohort ◽  
Gain Of Function ◽  
Transfected Cells ◽  
Aldosterone Production ◽  
H295r Cells

Abstract Most aldosterone-producing adenomas (APA) have gain-of-function somatic mutations of ion channels or transporters. However, their frequency in aldosterone-producing cell-clusters of normal adrenals suggests the existence of co-driver mutations which influence the development or phenotype of APAs. Gain-of-function mutations in both CTNNB1 and GNA11 were found by whole exome sequencing in 3 of 41 APAs from a UK/Irish cohort. Targeted sequencing for exon 3 mutations of CTNNB1 and p.Gln209 mutations of either GNA11 or closely homologous GNAQ confirmed these and 7 further double mutant APAs in this discovery cohort. The presence of GNA11/Q p.Gln209 mutations in CTNNB1 mutant APAs were replicated in 2 cohorts from France (n=14) and Sweden (n=3). In total, 16 (59%) of the 27 CTNNB1 mutant APAs investigated had a mutation at p.Gln209 of GNA11 (n=11) or GNAQ (n=5). Interestingly, CTNNB1-mutant APAs were more commonly present in women (23/27), and of these, those with GNA11/Q mutations were all women except for a pubertal boy. To also note, 9 of 10 of the UK/Irish double mutant APAs in the discovery cohort presented in puberty, pregnancy, or menopause. Mutation of p.Gln209, or homologous p.Gln in GNAS, GNA12-14, impair hydrogen bonds between G-protein α and β subunits. Transfection of H295R cells, an immortalised adrenocortical cell line heterozygous for the p.Ser45Pro mutation of CTNNB1 but wild-type for GNA11-14/Q/S, by each of the GNA11/Q mutations increased aldosterone secretion and CYP11B2 expression (encoding aldosterone synthase) by 1.93-6.1-fold and 8.0-9.8-fold respectively, compared to vector or wild-type -transfected cells. In ZG, GNA11/Q mediate the aldosterone response to angiotensin II, via stimulation of intracellular Ca2+ release by inositol trisphosphate. In the mutant-transfected cells, the stimulatory effect of angiotensin II 10 nM was retained. In order to determine whether the p.Gln209 mutations stimulate aldosterone production even in the absence of CTNNB1 activation, the transfections of H295R cells were repeated after either 24-h treatment with the CTNNB1 inhibitor, ICG-001, or silencing of CTNNB1 using the ONTARGETplus SMARTpool SiRNAs (Dharmacon). Both interventions reduced the aldosterone production relative to vehicle/control-treated cells; however neither ICG-001 nor silencing of CTNNB1 blunted the fold-increase in aldosterone secretion seen in mutant-transfected cells compared to wild-type. In summary, we report the discovery of gain-of-function mutations of the G-protein, GNA11, or its close homologue, GNAQ, in multiple APAs which majority presented during periods of high LH/HCG. To date, the mutation is always residue p.Gln209, and associated with a gain-of-function mutation of CTNNB1. These GNA11/Q p.Gln209 mutations increase aldosterone and CYP11B2 production both in the presence and in the absence of CTNNB1 activation.

scholarly journals A Cellular Assay for the Identification and Characterization of Connexin Gap Junction Modulators

2021 ◽  
Vol 22 (3) ◽  
pp. 1417
Author(s):  
Azeem Danish ◽  
Robin Gedschold ◽  
Sonja Hinz ◽  
Anke C. Schiedel ◽  
Dominik Thimm ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Small Molecules ◽  
High Throughput Screening ◽  
Cell Communication ◽  
Receptor Activation ◽  
Adenosine A2a Receptor ◽  
Intracellular Camp ◽  
Donor Cells ◽  
A2a Receptor ◽  
Adenosine A2a

Connexin gap junctions (Cx GJs) enable the passage of small molecules and ions between cells and are therefore important for cell-to-cell communication. Their dysfunction is associated with diseases, and small molecules acting as modulators of GJs may therefore be useful as therapeutic drugs. To identify GJ modulators, suitable assays are needed that allow compound screening. In the present study, we established a novel assay utilizing HeLa cells recombinantly expressing Cx43. Donor cells additionally expressing the Gs protein-coupled adenosine A2A receptor, and biosensor cells expressing a cAMP-sensitive GloSensor luciferase were established. Adenosine A2A receptor activation in the donor cells using a selective agonist results in intracellular cAMP production. The negatively charged cAMP migrates via the Cx43 gap junctions to the biosensor cells and can there be measured by the cAMP-dependent luminescence signal. Cx43 GJ modulators can be expected to impact the transfer of cAMP from the donor to the biosensor cells, since cAMP transit is only possible via GJs. The new assay was validated by testing the standard GJ inhibitor carbenoxolon, which showed a concentration-dependent inhibition of the signal and an IC50 value that was consistent with previously reported values. The assay was demonstrated to be suitable for high-throughput screening.

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