Angiotensin II induces coordinated calcium bursts in aldosterone-producing adrenal rosettes.
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ABSTRACT:
SUBMITTER: Guagliardo NA
PROVIDER: S-EPMC7125102 | biostudies-literature | 2020 Apr
REPOSITORIES: biostudies-literature
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Angiotensin II induces coordinated calcium bursts in aldosterone-producing adrenal rosettes.
Guagliardo Nick A NA Klein Peter M PM Gancayco Christina A CA Lu Adam A Leng Sining S Makarem Rany R RR Cho Chelsea C Rusin Craig G CG Breault David T DT Barrett Paula Q PQ Beenhakker Mark P MP
Nature communications 20200403 1
Aldosterone-producing zona glomerulosa (zG) cells of the adrenal gland arrange in distinct multi-cellular rosettes that provide a structural framework for adrenal cortex morphogenesis and plasticity. Whether this cyto-architecture also plays functional roles in signaling remains unexplored. To determine if structure informs function, we generated mice with zG-specific expression of GCaMP3 and imaged zG cells within their native rosette structure. Here we demonstrate that within the rosette, angi ...[more]
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| S-EPMC2666999 | biostudies-literature
Project description:The source of aldosterone in 30 to 40 % of patients with primary hyperaldosteronism (PA) is unilateral aldosterone-producing adenoma (APA). The mechanisms causing elevated aldosterone production in APA are unknown. Herein, we examined expression of G-protein coupled receptors (GPCR) in APA and demonstrate that compared to normal adrenals there is a general elevation of certain GPCR in many APA and/or ectopic expression of GPCR in others. RNA samples from normal adrenals (n = 5), APAs (n = 10), and cortisol-producing adenomas (CPAs) (n=13) were used on 15 genomic expression arrays, each of which included 223 GPCR transcripts presented in at least one out of 15 of the independent microarrays. The array results were confirmed using real-time RT-PCR (qPCR). Four GPCR transcripts exhibited a statistically significant increase that was greater than 3-fold compared to normal adrenals, suggesting a general increase in expression compared to normal adrenal glands. Four GPCR transcripts exhibited a greater than 15-fold increase of expression in one or more of the APA samples compared to normal adrenals. qPCR analysis confirmed array data and found the receptors with the highest fold increase in APA expression to be luteinizing hormone receptor (LH-R), serotonin receptor 4 (HTR4), gonadotropin-releasing hormone receptor (GnRHR), glutamate receptor metabotropic 3 (GRM3), endothelin receptor type B-like protein (GPR37), and ACTH receptor (MC2R). There are also sporadic increased expressions of these genes in the CPAs. Together, these findings suggest a potential role of altered GPCR expression in many cases of PA and provide candidate GPCR for further study. Keywords: disease state analysis Normal human adult adrenals were obtained through the Cooperative Human Tissue Network (Philadelphia, PA) and Clontech (Palo Alto, CA). All the normal adrenal samples came from patients that underwent adrenalectomy along with a renalectomy due to renal carcinoma. The adrenal samples acquired from autopsies were obtained no more than six hours after death, and it was confirmed that the causes of death were unrelated to adrenal function. Real-time quantitative RT-PCR was used to check the legitimacy of the normal control adrenal samples. Examination of tissue sections from each adrenal gland also suggested normal histology. The APA samples were obtained from the Departments of Medicine at UT Southwestern and Division of Endocrinology at the University of Padua. All APA samples were from Conn’s Syndrome patients with significantly elevated circulating aldosterone that was lowered to the normal range after surgical removal of the tumor. Thirty-two adenomas were collected from patients with primary hyperaldosteronism. Twenty-eight of these adenomas had levels of CYP11B2 that were two standard deviations (SD) greater than seen in normal adrenal glands and these samples were used for analyses. It is currently not clear if the adenomas with low CYP11B2 expression are the source of aldosterone in these patients. Other studies suggest that subcapsular micronodules have varying expression of steroidogenic enzymes-some with aldosterone synthase and some without (Shigematsu et al., 2006). Future studies are required to determine if some tumor-bearing adrenals from primary hyperaldosteronsim patients also have small CYP11B2 positive micronodules as the source of aldosterone production. The cortisol-producing adenoma (CPA) tissues were obtained from Division of Endocrinology at the University of Padua from patients with Cushing’s syndrome. Each of the patients was cured following surgical removal of the adenoma. The use of these tissues was approved by the Institutional Review Boards of the University of Texas Southwestern Medical Center (Dallas, TX), the Medical College of Georgia (Augusta, GA), and University of Padua (Italy). In addition, this study was approved by the ethical committee at the University of Padua, and informed consent was obtained from every patient. Microarray Analysis Total RNA isolated from normal adult adrenal glands (n = 5) and APA (n = 10) were used on 15 genomic expression arrays. In brief, RNA was hybridized to an Affymetrix human HG-U133+2 oligonucleotide microarray set containing 54,675 probe sets representing approximately 40,500 independent human genes. The arrays were scanned at high resolution using an Affymetrix GeneChip Scanner 3000 located at Medical College of Georgia Microarray Core Facility (Augusta, GA). Results were analyzed using GeneSpring GX 7.3.1 software (Silicon Genetics, Redwood City, CA) to identify differences in expression of GPCR between normal adult adrenal and APA. RNA Extraction The tissue was pulverized in liquid nitrogen to a powder. Total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA). The purity and integrity of the RNA were assessed by the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) and its quantity was determined by NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE). cDNA Synthesis 2 µg of total RNA was reverse transcribed using the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA) following manufacturer recommendations and incubated at 25 ºC for 10 min, and 37 ºC for 2 h. The synthesized cDNA was subjected to 1:10 dilution and stored at -20ºC. Real-Time Quantitative PCR Primers and probes for the amplification of the selected GPCR sequences were performed using the TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA) based on published sequences for genes encoding the respective human GPCR. The gene symbols and AB assay numbers are listed in Table 1. The primer and probe set for human CYP11B2 was designed using Primer Express 3.0 (Applied Biosystems, Foster City, CA) and purchased from IDT (Integrated DNA Technologies, Inc, IA) as published previously (Saner-Amigh et al., 2006). In brief: Forward: 5’-GGCAGAGGCAGAGATGCTG-3’, Reverse: 5’- CTTGAGTTAGTGTCTCCACCAGGA-3’, Probe: 5’-CTGCACCACGTGCTGAAGCACT-3’. PCR reactions were performed using the ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA) with a total volume of 20 μl per reaction following the reaction parameters recommended by the manufacturer, which includes denaturation at 95ºC for 20 s followed by amplification for 40 cycles (95ºC at 3 s, 60ºC at 30 s, fluorescence measurement). For each GPCR the 20 μl total volume consisted of 10 µl TaqMan Fast Universal PCR Master Mix (2X) (Applied Biosystems, Foster City, CA), 900 nM of each primer, 400 nM of the probe and 5 μl of each first-strand cDNA sample. CYP11B2 reaction mix consisted of 10µl TaqMan PCR Master Mix (2X) (Applied Biosystems, Foster City, CA), 100 nM of primer/probe mix and 5 μl of each first-strand cDNA sample. 18s rRNA was detected and quantified using the TaqMan Ribosomal RNA Control Reagents (Vic Probe) (Applied Biosystems, Foster City, CA). Each reaction included 10 µl of TaqMan PCR Master Mix (2X) (Applied Biosystems, Foster City, CA), 100 nM probe and 50 nM primers. Negative controls contained water instead of first-strand cDNA. Quantitative normalization of cDNA in each tissue-derived sample was performed using expression of 18s rRNA as an internal control. The generated Ct value of each gene was normalized by its respected Ct value of 18s rRNA (∆Ct). Then each gene was further normalized using the average ∆Ct value of the normal adult adrenal (∆∆Ct). The final fold-expression changes were calculated using the equation 2-∆∆Ct (Livak & Schmittgen, 2001). Statistical Analysis Data were analyzed and compared to control values (mean of normal adrenal samples) using the Mann-Whitney Rank Sum test with the SigmaStat 3.0 software package (SPSS, Chicago, IL). Results were considered significantly different when p value was 0.05.
2007-07-18 | E-GEOD-8514 | biostudies-arrayexpress
Project description:The source of aldosterone in 30 to 40 % of patients with primary hyperaldosteronism (PA) is unilateral aldosterone-producing adenoma (APA). The mechanisms causing elevated aldosterone production in APA are unknown. Herein, we examined expression of G-protein coupled receptors (GPCR) in APA and demonstrate that compared to normal adrenals there is a general elevation of certain GPCR in many APA and/or ectopic expression of GPCR in others. RNA samples from normal adrenals (n = 5), APAs (n = 10), and cortisol-producing adenomas (CPAs) (n=13) were used on 15 genomic expression arrays, each of which included 223 GPCR transcripts presented in at least one out of 15 of the independent microarrays. The array results were confirmed using real-time RT-PCR (qPCR). Four GPCR transcripts exhibited a statistically significant increase that was greater than 3-fold compared to normal adrenals, suggesting a general increase in expression compared to normal adrenal glands. Four GPCR transcripts exhibited a greater than 15-fold increase of expression in one or more of the APA samples compared to normal adrenals. qPCR analysis confirmed array data and found the receptors with the highest fold increase in APA expression to be luteinizing hormone receptor (LH-R), serotonin receptor 4 (HTR4), gonadotropin-releasing hormone receptor (GnRHR), glutamate receptor metabotropic 3 (GRM3), endothelin receptor type B-like protein (GPR37), and ACTH receptor (MC2R). There are also sporadic increased expressions of these genes in the CPAs. Together, these findings suggest a potential role of altered GPCR expression in many cases of PA and provide candidate GPCR for further study. Keywords: disease state analysis
2007-07-19 | GSE8514 | GEO
Project description:To investigate the development of aldosterone-producing adenoma, we profiled cells of different areas of the adjacent adrenal cortex by snRNAseq to obtain possible trajectories towards adenoma formation.
2022-08-05 | GSE210381 | GEO
Project description:BackgroundThe Renin-Angiotensin-Aldosterone-System plays a pivotal role in hypertension. Angiotensin II (Ang II) is a major regulator of aldosterone synthesis and secretion, and it is known to facilitate reactive oxygen species (ROS) generation in many cell types.AimsHere, we assessed the role of ROS signaling in Ang II-induced aldosterone synthesis by focusing on the regulation of aldosterone synthase (CYP11B2), a cytochrome P450 oxidase that catalyzes the final step in aldosterone biosynthetic pathway.ResultsAng II increased CYP11B2 activity, mRNA and protein with a concomitant elevation of 6-Carboxy- 2',7'-dichlorodihydrofluorescein diacetate fluorescence, malondialdehyde and protein carbonyl levels (indices of ROS), NADPH oxidase (Nox) activity, and H(2)O(2) levels in human and rat adrenal cortical cells. The expression of nuclear receptor related 1 protein, a transcription factor known to regulate CYP11B2 expression, was also augmented by Ang II. These Ang II-evoked effects were either abolished or attenuated by pretreatment of cells with either Ang II type I receptor (AT(1)R) antagonist, or antioxidants or Nox inhibitor or siRNA silencing of Nox1, 2 and 4, or inhibitors of phospholipase C and protein kinase C. Exogenous H(2)O(2) mimicked the facilitatory effects of Ang II on CYP11B2 activity, mRNA, and protein expression, and these changes were significantly reduced by PEG-catalase.InnovationROS, particularly H(2)O(2), is identified as a key regulator of aldosterone production.ConclusionOur results suggest that Ang II facilitates CYP11B2 activity and the ensuing aldosterone production via activation of AT(1)R-Nox-H(2)O(2) signaling pathway.
| S-EPMC3365360 | biostudies-literature
Project description:The renin-angiotensin-aldosterone system has an important role in the control of fluid homeostasis and BP during volume depletion. Dietary salt restriction elevates circulating angiotensin II (AngII) and aldosterone levels, increasing levels of the Cl-/HCO3- exchanger pendrin in β-intercalated cells and the Na+-Cl- cotransporter (NCC) in distal convoluted tubules. However, the independent roles of AngII and aldosterone in regulating these levels remain unclear. In C57BL/6J mice receiving a low-salt diet or AngII infusion, we evaluated the membrane protein abundance of pendrin and NCC; assessed the phosphorylation of the mineralocorticoid receptor, which selectively inhibits aldosterone binding in intercalated cells; and measured BP by radiotelemetry in pendrin-knockout and wild-type mice. A low-salt diet or AngII infusion upregulated NCC and pendrin levels, decreased the phosphorylation of mineralocorticoid receptor in β-intercalated cells, and increased plasma aldosterone levels. Notably, a low-salt diet did not alter BP in wild-type mice, but significantly decreased BP in pendrin-knockout mice. To dissect the roles of AngII and aldosterone, we performed adrenalectomies in mice to remove aldosterone from the circulation. In adrenalectomized mice, AngII infusion again upregulated NCC expression, but did not affect pendrin expression despite the decreased phosphorylation of mineralocorticoid receptor. By contrast, AngII and aldosterone coadministration markedly elevated pendrin levels in adrenalectomized mice. Our results indicate that aldosterone is necessary for AngII-induced pendrin upregulation, and suggest that pendrin contributes to the maintenance of normal BP in cooperation with NCC during activation of the renin-angiotensin-aldosterone system by dietary salt restriction.
| S-EPMC5748905 | biostudies-literature