The angiotensin II-AT1 receptor stimulates reactive oxygen species within the cell nucleus

Abstract

We and others have reported significant expression of the Ang II Type 1 receptor (AT1R) on renal nuclei; thus, the present study assessed the functional pathways and distribution of the intracellular AT1R on isolated nuclei. Ang II (1nM) stimulated DCF fluorescence, an intranuclear indicator of reactive oxygen species (ROS), while the AT1R antagonist losartan or the NADPH oxidase (NOX) inhibitor DPI abolished the increase in ROS. Dual labeling of nuclei with antibodies against nucleoporin 62 (Nup62) and AT1R or the NADPH oxidase isoform NOX4 revealed complete overlap of the Nup62 and AT1R (99%) by flow cytometry, while NOX4 was present on 65% of nuclei. Treatment of nuclei with a PKC agonist increased ROS while the PKC inhibitor GF109203X or PI3 kinase inhibitor LY294002 abolished Ang II stimulation of ROS. We conclude that the Ang II-AT1R-PKC axis may directly influence nuclear function within the kidney through a redox sensitive pathway.

Figures

Figure 1

Angiotensin (Ang) II stimulates reactive oxygen species (ROS) in renal cortical nuclei. ROS was visualized with dichlorofluroscein (DCF, 20 μM) in isolated renal cortical nuclei by flow cytometry. Nuclei were incubated with Ang II (1 nM) for 5 min at 37°C. Inhibitors were added 10 min prior to Ang II-stimulated or control nuclei. Panel A: Representative fluorescence tracings of Ang II (1 nM) as compared to control. Panel B: Ang II and the AT1 receptor antagonist losartan (Los, 10 μM); Panel C: Ang II and the NADPH oxidase inhibitor DPI (10 μM). Panel D: Ang II and the combination of LOS and DPI. Panel E: Changes in mean fluorescence intensity (% MFI) as compared to control nuclei. Data are means + SE from 4 – 6 experiments, *P

Figure 2

AT1 receptor and NOX4 localization in renal cortical nuclei by flow cytometry. Panel A: Nuclear labeling with the AT1 receptor and nucleoporin 62 (Nup62) antibodies. Panel B: Nuclear labeling with the isotype antibody controls for AT1 receptor (rabbit IgG-labeled phycoerythrin, PE) and Nup62 (mouse IgG-labeled fluorosceinisothyocyanate, FITC). Panel C: Immunoblot of 3 separate renal nuclear preparations with the AT1 receptor antibody. Panel D: NOX4 antibody labeling with Nup62. Panel E: Nuclear labeling with the isotype antibody controls for NOX4 receptor (rabbit IgG-PE) and Nup62 (mouse IgG-FITC). Panel F: Immunoblot of 4 separate renal nuclear preparations with the NOX4 antibody. Molecular weight (MW) expressed in kilodaltons (kDa).

Figure 3

Angiotensin (Ang) II-dependent reactive oxygen species (ROS) production is sensitive to PKC and PI3 kinase inhibitors. ROS was visualized with dichlorofluroscein (DCF, 20 μM) in isolated renal cortical nuclei by flow cytometry. Nuclei were incubated with Ang II (1 nM) for 5 min at 37°C. Inhibitors were added 10 min prior to Ang II-stimulated or control nuclei. Panel A: Representative fluorescence tracing of the PKC activator PMA (1 μM) compared to control. Panel B: Ang II (1 nM) and the PKC inhibitor GF 109203X (GF, 500 nM). Panel C: Ang II and the PI3 kinase inhibitor LY 294002 (LY, 10 μM). Panel D: Changes in mean fluorescence intensity (% MFI) as compared to control nuclei. Data are means + SE from 3 – 6 experiments, *P