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.

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

Mechanisms of ET-1 potentiation of angiotensin II stimulation of aldosterone production

1993 ◽  
Vol 265 (2) ◽  
pp. E179-E183 ◽  
Author(s):  
E. N. Cozza ◽  
C. E. Gomez-Sanchez
Keyword(s):  
Angiotensin Ii ◽  
Zona Glomerulosa ◽  
Ang Ii ◽  
Aldosterone Secretion ◽  
Direct Stimulation ◽  
Potentiation Effect ◽  
Aldosterone Production ◽  
Glomerulosa Cells ◽  
Pkc Inhibitors ◽  
Stimulation Of

Endothelin-1 (ET-1) exerts the following two types of aldosterone-stimulating actions on glomerulosa cells: ET-1-mediated direct stimulation of aldosterone secretion (per se effect) and potentiation of the aldosterone secretion to angiotensin II (ANG II; potentiation effect). The role of Ca2+ and protein kinase C (PKC) systems in these two effects was investigated. Incubations of calf cultured adrenal zona glomerulosa cells in low-Ca2+ media or in the presence of the Ca2+ channel antagonist verapamil reduced the aldosterone secretion to ET-1. When cells were preincubated with ET-1 in a low-Ca2+ media or in the presence of the Ca2+ channel antagonist verapamil, washed, and incubated in media with normal Ca2+, ANG II showed potentiation of ANG II-stimulated aldosterone secretion. The PKC inhibitors H-7 and staurosporine did not decrease ET-1-stimulated aldosterone secretion, but they inhibited the potentiation effect of ET-1 on ANG II-mediated aldosterone secretion. Adrenocorticotropic hormone desensitization or prolonged phorbol ester stimulation of PKC resulting in desensitization also resulted in the abolition of the ET-1-mediated ANG II potentiation of aldosterone secretion. The PKC inhibitors did not affect ANG II-stimulated aldosterone secretion. We conclude that ET-1 exerts a direct stimulation of aldosterone secretion through a mechanism dependent on Ca2+ and potentiates ANG II-mediated aldosterone stimulation through a mechanism involving PKC.

scholarly journals Steroidogenesis in Aldosterone-Producing Adenoma Revisited by Transcriptome Analysis

2005 ◽  
Vol 90 (12) ◽  
pp. 6638-6649 ◽  
Author(s):  
Guillaume Assié ◽  
Colette Auzan ◽  
Jean-Marie Gasc ◽  
Erno Baviera ◽  
André Balaton ◽  
...  
Keyword(s):  
Gene Expression ◽  
Angiotensin Ii ◽  
Calcium Binding ◽  
Density Lipoprotein ◽  
Zona Glomerulosa ◽  
Differentially Expressed ◽  
Calcium Activation ◽  
Starting Point ◽  
Aldosterone Production ◽  
Aldosterone Producing Adenoma

Context: Primary aldosteronism (PAL) is the most frequent cause of secondary arterial hypertension. In PAL, aldosterone production is chronic, excessive, and autonomous. Objective: The objective of this study was to identify the angiotensin-II independent alterations of steroidogenesis responsible for PAL. Design: Genomewide gene expression was compared in two tissues differentiated for aldosterone production, both nonstimulated by circulating angiotensin II and differing in their autonomy to produce aldosterone: aldosterone-producing adenoma (APA) and its adjacent dissected zona glomerulosa (ZG). Setting: The setting of this study was the Comete Network. Patients: Patients with APA were studied. Intervention: Transcriptome comparison was made of one APA and its adjacent ZG by serial analysis of gene expression; validation by in situ hybridization was performed for 19 genes in 11 samples. Outcome: The study outcome was genes differentially expressed in APA and adjacent ZG. Results: Activation of steroidogenesis in PAL is restricted to the overexpression of the enzymes producing aldosterone-specific steroids, aldosterone synthase and also 21-hydroxylase, suggesting that upstream precursor production is not limiting. Increased expression of high-density lipoprotein receptor, adrenodoxin and P450 oxidoreductase suggests that these systems provide cholesterol and electrons to the mitochondrial steroidogenic enzymes. As for acute stimulation of aldosterone production, an activation of calcium signaling is suggested by concordant overexpression of calcium-binding proteins or effectors. Calcium activation may result from an abnormal activity of Gq protein-coupled receptors. This calcium activation may be the starting point of the other gene expression changes observed in APA. Finally, other differentially expressed genes include three genes encoding unidentified proteins. Conclusion: This work provides an original and integrated view of the mechanisms of aldosterone production in PAL.

Regulation of aldosterone production from zona glomerulosa cells by ANG II and cAMP: evidence for PKA-independent activation of CaMK by cAMP

2006 ◽  
Vol 290 (3) ◽  
pp. E423-E433 ◽  
Author(s):  
Stepan Gambaryan ◽  
Elke Butt ◽  
Piet Tas ◽  
Albert Smolenski ◽  
Bruno Allolio ◽  
...  
Keyword(s):  
Intracellular Signaling ◽  
Mapk Signaling ◽  
Zona Glomerulosa ◽  
Intracellular Camp ◽  
Ang Ii ◽  
Intact Cells ◽  
Molecular Events ◽  
Aldosterone Production ◽  
Dependent Protein

Aldosterone production in zona glomerulosa (ZG) cells of adrenal glands is regulated by various extracellular stimuli (K+, ANG II, ACTH) that all converge on two major intracellular signaling pathways: an increase in cAMP production and calcium (Ca2+) mobilization. However, molecular events downstream of the increase in intracellular cAMP and Ca2+ content are controversial and far from being completely resolved. Here, we found that Ca2+/calmodulin-dependent protein kinases (CaMKs) play a predominant role in the regulation of aldosterone production stimulated by ANG II, ACTH, and cAMP. The specific CaMK inhibitor KN93 strongly reduced ANG II-, ACTH-, and cAMP-stimulated aldosterone production. In in vitro kinase assays and intact cells, we could show that cAMP-induced activation of CaMK, using the adenylate cyclase activator forskolin or the cAMP-analog Sp-5,6-DCI-cBIMPS (cBIMPS), was not mediated by PKA. Activation of the recently identified cAMP target protein Epac (exchange protein directly activated by cAMP) by 8-pCPT-2′- O-Me-cAMP had no effect on CaMK activity and aldosterone production. Furthermore, we provide evidence that cAMP effects in ZG cells do not involve Ca2+ or MAPK signaling. Our results suggest that ZG cells, in addition to PKA and Epac/Rap proteins, contain other as yet unidentified cAMP mediator(s) involved in regulating CaMK activity and aldosterone secretion.

scholarly journals Regulators of G-protein signaling 4 in adrenal gland: localization, regulation, and role in aldosterone secretion

2007 ◽  
Vol 194 (2) ◽  
pp. 429-440 ◽  
Author(s):  
Damian G Romero ◽  
Ming Yi Zhou ◽  
Licy L Yanes ◽  
Maria W Plonczynski ◽  
Tanganika R Washington ◽  
...  
Keyword(s):  
Adrenal Gland ◽  
G Protein ◽  
Intracellular Signaling ◽  
Zona Glomerulosa ◽  
G Protein Signaling ◽  
Ang Ii ◽  
Protein Signaling ◽  
Aldosterone Secretion ◽  
H295r Cells

Regulators of G-protein signaling (RGS proteins) interact with Gα subunits of heterotrimeric G-proteins, accelerating the rate of GTP hydrolysis and finalizing the intracellular signaling triggered by the G-protein-coupled receptor (GPCR)–ligand interaction. Angiotensin II (Ang II) interacts with its GPCR in adrenal zona glomerulosa cells and triggers a cascade of intracellular signals that regulates steroidogenesis and proliferation. On screening for adrenal zona glomerulosa-specific genes, we found that RGS4 was exclusively localized in the zona glomerulosa of the rat adrenal cortex. We studied RGS4 expression and regulation in the rat adrenal gland, including the signaling pathways involved, as well as the role of RGS4 in steroidogenesis in human adrenocortical H295R cells. We reported that RGS4 mRNA expression in the rat adrenal gland was restricted to the adrenal zonal glomerulosa and upregulated by low-salt diet and Ang II infusion in rat adrenal glands in vivo. In H295R cells, Ang II caused a rapid and transient increase in RGS4 mRNA levels mediated by the calcium/calmodulin/calmodulin-dependent protein kinase and protein kinase C pathways. RGS4 overexpression by retroviral infection in H295R cells decreased Ang II-stimulated aldosterone secretion. In reporter assays, RGS4 decreased Ang II-mediated aldosterone synthase upregulation. In summary, RGS4 is an adrenal gland zona glomerulosa-specific gene that is upregulated by aldosterone secretagogues, in vivo and in vitro, and functions as a negative feedback of Ang II-triggered intracellular signaling. Alterations in RGS4 expression levels or functions may be involved in deregulations of Ang II signaling and abnormal aldosterone secretion.

Endothelin-1 potentiation of angiotensin II stimulation of aldosterone production

1992 ◽  
Vol 262 (1) ◽  
pp. R85-R89 ◽  
Author(s):  
E. N. Cozza ◽  
S. Chiou ◽  
C. E. Gomez-Sanchez
Keyword(s):  
Angiotensin Ii ◽  
Zona Glomerulosa ◽  
Receptor Subtype ◽  
Ang Ii ◽  
Aldosterone Secretion ◽  
Acth Stimulation ◽  
Endothelin 1 ◽  
Aldosterone Production ◽  
Glomerulosa Cells ◽  
Stimulation Of

Endothelin-1 (ET-1) binds to specific receptors in cultured bovine adrenal glomerulosa cells and stimulates aldosterone secretion with a 50% effective concentration (EC50) of 300 +/- 80 pM (mean +/- SE). The relative stimulatory potency for ET-1 is significantly less than that of angiotensin II (ANG II). The incubation of calf zona glomerulosa cells in primary culture with ET-1 and ANG II resulted in a significant potentiation of ANG II effect on aldosterone secretion. The EC50 of ET-1 potentiation of ANG II-induced stimulation of aldosterone secretion was 40 +/- 5 pM (mean +/- SE, n = 4), which is lower than the EC50 for ET-1 stimulation of aldosterone secretion. Adrenocorticotropic hormone (ACTH) stimulation of aldosterone secretion, but not that of potassium, was also potentiated by ET-1, but to a lesser degree. ET-1 and ET-1-mediated potentiation of ANG II-stimulated aldosterone biosynthesis increased both the early and late pathways of aldosterone biosynthesis, but the potentiation was greater for the early pathway. Preincubation with ET-1 for at least 15 min, followed by extensive washing to remove bound ET-1, also resulted in persistent potentiation of ANG II-mediated aldosterone secretion. ET-2, sarafotoxin, and vasoactive intestinal contractor potentiation of ANG II action were very similar to that of ET-1. ET-3 and Big-ET-1 potentiated ANG II stimulation only at the highest doses tested and the proendothelin-(110-130) fragment was inactive. ET-1 potentiation of ANG II action is likely to be mediated through an ETB receptor subtype.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Rate of calcium entry determines the rapid changes in protein kinase C activity in angiotensin II-stimulated adrenal glomerulosa cells

1994 ◽  
Vol 297 (3) ◽  
pp. 523-528 ◽  
Author(s):  
I Kojima ◽  
N Kawamura ◽  
H Shibata
Keyword(s):  
Protein Kinase C ◽  
Angiotensin Ii ◽  
Protein Kinase ◽  
Specific Substrate ◽  
Ang Ii ◽  
Kinase C ◽  
Aldosterone Production ◽  
Glomerulosa Cells ◽  
Peptide Phosphorylation

The present study was conducted to monitor precisely the activity of protein kinase C (PKC) in adrenal glomerulosa cells stimulated by angiotensin II (ANG II). PKC activity in cells was monitored by measuring phosphorylation of a synthetic KRTLRR peptide, a specific substrate for PKC, immediately after the permeabilization of the cells with digitonin [Heasley and Johnson J. Biol. Chem. (1989) 264, 8646-8652]. Addition of 1 nM ANG II induced a gradual increase in KRTLRR peptide phosphorylation, which reached a peak at 30 min, and phosphorylation was sustained thereafter. When the action of ANG II was terminated by adding [Sar1,Ala8]ANG II, a competitive antagonist, both Ca2+ entry and KRTLRR phosphorylation ceased rapidly, whereas diacylglyercol (DAG) content was not changed significantly within 10 min. Similarly, when blockade of Ca2+ entry was achieved by decreasing extracellular Ca2+ to 1 microM or by adding 1 microM nitrendipine, KRTLRR peptide phosphorylation was decreased within 5 min. In addition, restoration of Ca2+ entry was accompanied by an immediate increase in KRTLRR peptide phosphorylation. Under the same condition, DAG content did not change significantly. We then examined the role of the PKC pathway in ANG II-induced aldosterone production. Ro 31-8220 inhibited ANG II-induced KRTLRR phosphorylation without affecting the activity of calmodulin-dependent protein kinase II. In the presence of Ro 31-8220, ANG II-mediated aldosterone production was decreased to approx. 50%. Likewise, intracellular administration of PKC19-36, a sequence corresponding to residues 19-36 of the regulatory domain of PKC known to inhibit PKC activity, attenuated ANG II-mediated activation of PKC and aldosterone output. These results indicate a critical role of Ca2+ entry in the regulation of PKC activity by ANG II.

Effect of CO2/pH on the aldosterone response to hypoxia in bovine adrenal cells in vitro

1993 ◽  
Vol 265 (4) ◽  
pp. R820-R825
Author(s):  
H. Raff ◽  
B. Jankowski
Keyword(s):  
Respiratory Acidosis ◽  
Inhibitory Effect ◽  
Zona Glomerulosa ◽  
Ang Ii ◽  
Adrenal Cells ◽  
Aldosterone Production ◽  
Per Se ◽  
Air Control

Acidosis increases and hypoxia decreases aldosterone production from the adrenal zona glomulerosa in vivo, in situ, and in vitro. These effects appear to be located at different steps in the steroidogenic process. Because respiratory acidosis and hypoxemia are common sequelae of chronic lung disease, the present experiments evaluated the interaction of hypoxia and CO2 (with uncompensated or compensated extracellular pH) on aldosteronogenesis in vitro. Bovine adrenal zona glomerulosa cells were stimulated with angiotensin II (ANG II) or adenosine 3',5'-cyclic monophosphate under room air control (21% O2-0% CO2), CO2 per se (21% O2-10% CO2), hypoxia per se (10% O2-0% CO2), and the combination of CO2 and hypoxia (10% O2-10% CO2). Furthermore, under CO2, pH was either allowed to decrease from 7.2 to 6.8 (uncompensated) or its decrease was minimized (> 7.05) with NaOH (compensated). CO2 without pH compensation led to a significant increase in ANG II-stimulated aldosterone release; when the decrease in pH was minimized, CO2 inhibited ANG II-stimulated aldosterone release. Hypoxia inhibited aldosterone release; the inhibitory effect of hypoxia predominated when combined with CO2. In the presence of cyanoketone, pregnenolone production from endogenous precursors (early pathway) was unaffected. However, the conversion of corticosterone to aldosterone (late pathway) was inhibited by low O2 but unaffected by CO2. It is concluded that the inhibitory effect of low O2 on the late pathway predominates over the effects of uncompensated or compensated simulated respiratory acidosis on aldosteronogenesis.

Angiotensin II-dependent proximal tubule sodium transport requires receptor-mediated endocytosis

1994 ◽  
Vol 266 (3) ◽  
pp. C669-C675 ◽  
Author(s):  
J. R. Schelling ◽  
S. L. Linas
Keyword(s):  
Angiotensin Ii ◽  
Proximal Tubule ◽  
Receptor Internalization ◽  
Ang Ii ◽  
Na Transport ◽  
Cellular Mechanisms ◽  
Transport Pathways ◽  
Proximal Tubule Cells ◽  
Receptor Mediated Endocytosis ◽  
Tubule Cells

Angiotensin II (ANG II) receptors are present on apical and basolateral surfaces of proximal tubule cells. To determine the cellular mechanisms of proximal tubule ANG II receptor-mediated Na transport, apical-to-basolateral 22Na flux was measured in cultured proximal tubule cells. Apical ANG II caused increases in 22Na flux (maximum response: 100 nM, 30 min). Basolateral ANG II resulted in 22Na flux that was 23-56% greater than 22Na flux observed with equimolar apical ANG II. Apical ANG II-induced 22Na flux was prevented by preincubation with amiloride, ouabain, and the AT1 receptor antagonist losartan. Because apical ANG II signaling was previously shown to be endocytosis dependent, we questioned whether endocytosis was required for ANG II-stimulated proximal tubule Na transport as well. Apical (but not basolateral) ANG II-dependent 22Na flux was inhibited by phenylarsine oxide, an agent which prevents ANG II receptor internalization. In conclusion, apical and basolateral ANG II caused proximal tubule Na transport. Apical ANG II-dependent Na flux was mediated by AT1 receptors, transcellular transport pathways, and receptor-mediated endocytosis.

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