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GPER Attenuates Angiotensin II‐Induced Oxidative Stress via cAMP‐Mediated Regulation of NOX4
Author(s) -
OGOLA BENARD O.,
Zimmerman Margaret A.,
Sure Venkata N.,
Katakam Prasad V.G.,
Lindsey Sarah H.
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.700.1
Subject(s) - gper , oxidative stress , angiotensin ii , medicine , endocrinology , chemistry , nadph oxidase , reactive oxygen species , nox4 , agonist , p22phox , phenylephrine , receptor , estrogen receptor , biochemistry , biology , cancer , breast cancer , blood pressure
Our previous work shows that the G protein‐coupled estrogen receptor (GPER) is protective in the vasculature and kidneys during angiotensin (Ang) II‐dependent hypertension, in part by inhibiting oxidative stress. In addition, we find that acute estrogenic signaling via GPER involves activation of the cAMP signaling pathway in vascular smooth muscle cells. Ang II stimulates the production of reactive oxygen species (ROS) via NADPH oxidase (NOX) complex activation, but whether GPER interferes with NOX activation and whether this occurs through cAMP signaling is not known. Therefore, the goal of the current study was to assess the molecular mechanisms by which GPER attenuates Ang II‐induced oxidative stress in vitro . A7r5 rat aortic smooth muscle cells were treated overnight with 100 nM Ang II in the presence of vehicle or 100 nM of the GPER agonist G‐1. ROS was measured via electron spin resonance spectroscopy (e‐scan) and CellRox™ Green fluorescence. NOX activity and expression were measured by NADP/NADPH ratio, real time RT‐PCR, and immunoblotting. Both e‐scan and CellRox experiments showed that Ang II increased oxidative stress, while co‐administration of the GPER agonist G‐1 prevented this increase (4.3 × 10 5 vs 1.9 × 10 5 AU/mg protein, P <0.001, n=7–8). Similarly, NOX activity was elevated in response to Ang II but normalized by G‐1 (189% vs. 83% RLU, P <0.0001, n=5). When the production of cAMP was inhibited with the adenylyl cyclase inhibitor SQ22536, G‐1 was unable to block Ang II‐induced NOX activity (153% vs. 142% RLU, P= 0.39, n=6). Ang II increased NOX4 mRNA over time, with a maximum increase at 4h (3.5‐fold change, P <0.05, n=3). Ang II also increased NOX4 protein (1.2 vs 2.1 ADU/β‐actin, P< 0.01, n=2). In contrast, administration of G‐1 for 4h decreased NOX4 mRNA by 60% ( P< 0.05, n=3) with no significant impact on protein expression ( P =0.91, n=2). Preliminary studies using male wildtype and knockout mice show higher expression of NOX4 in response to genetic deletion of GPER. These experiments indicate that GPER inhibits NOX4 transcription and suppresses NOX activity, thereby limiting the production of ROS by Ang II. The impact of GPER on NOX activity was dependent on the production of cAMP. Taken together with our previous work, this study provides insight into how acute estrogen signaling provides cardiovascular protection during Ang II‐induced hypertension and potentially other diseases that are characterized by increased oxidative stress. Support or Funding Information This study was funded by National Institutes of Health grant HL133619 to S.H.L. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .