Identification and characterization of a functional mitochondrial angiotensin system

Abstract

The renin-angiotensin (Ang) system regulates multiple physiological functions through Ang II type 1 and type 2 receptors. Prior studies suggest an intracellular pool of Ang II that may be released in an autocrine manner upon stretch to activate surface membrane Ang receptors. Alternatively, an intracellular renin-Ang system has been proposed, with a primary focus on nuclear Ang receptors. A mitochondrial Ang system has not been previously described. Here we report that functional Ang II type 2 receptors are present on mitochondrial inner membranes and are colocalized with endogenous Ang. We demonstrate that activation of the mitochondrial Ang system is coupled to mitochondrial nitric oxide production and can modulate respiration. In addition, we present evidence of age-related changes in mitochondrial Ang receptor expression, i.e., increased mitochondrial Ang II type 1 receptor and decreased type 2 receptor density that is reversed by chronic treatment with the Ang II type 1 receptor blocker losartan. The presence of a functional Ang system in human mitochondria provides a foundation for understanding the interaction between mitochondria and chronic disease states and reveals potential therapeutic targets for optimizing mitochondrial function and decreasing chronic disease burden with aging.

Fig. 1.

Immuno-electron microscopic localization of AT₁Rs in sections of human monocyte (A and B) and mouse kidney tubular cells (C and D) using gold bead labeling (arrows) for AT₁Rs. A shows AT1Rs on human monocyte cell membrane and in the cytoplasm (B). C shows labeling in close proximity to mitochondria and rarely in the mitochondria (D).

Fig. 2.

Immuno-electron microscopic localization of AT₂Rs in sections of human monocyte (A and B) and mouse renal tubular cells (C and D) using gold beads labeling (arrows) for AT₂Rs. A shows AT₂Rs on human monocyte cell membrane, and B–D reveal heavy labeling for AT₂Rs within mitochondria.

Fig. 3.

Immunoelectron microscopic localization of AT₂R binding to Ang in the mitochondria by using a gold-labeled anti-AT₂R antibody (12 nm gold) and a gold-labeled anti-Ang antibody (6 nm gold). Shown is colocalization of AT₂Rs with Ang in sections of mouse hepatocytes (A), kidney tubular cells (B), neurons (C), and cardiac myocytes (D).

Fig. 4.

Transfected AT₂Rs colocalize with mitochondria in human fibroblasts. Human fibroblast cells were transfected with pcDNA-Cycle 3 GFP-AT₂R construct (B) or positive control using pcDNA-EGFP-C1 (F) and counterstained with MitoTracker Red (A and E) (100× oil immersion). The merged images show yellow fluorescence (C and G). Fluorographic analysis (D and H) reveals a high correlation coefficient (R² = 0.72), suggesting a strong colocalization between AT₂Rs and MitoTracker within the mitochondria.

Fig. 5.

Purification of inner mitochondrial membrane AT₂Rs. (A) Whole-liver homogenate fractionations up to the inner mitochondrial membrane were subjected to 12% SDS/PAGE and immunoblotting with anti-AT₂R as well as anti-Na⁺/K⁺ ATPase, anti-VDAC, and anti-ATP synthase β for detecting cell membrane, outer mitochondrial membrane, and inner mitochondrial membrane markers, respectively. AT₂Rs tracked with inner mitochondrial membrane marker ATP synthase β, consistent with inner mitochondrial membrane localization of AT₂Rs. (B) Integrated densitometric band analysis of immunoblots demonstrating fold enrichment of AT₂Rs, inner mitochondrial membrane marker (ATP synthase), outer mitochondrial membrane marker (VDAC), and plasma membrane marker (Na/K ATPase) with mitochondrial purification. (C) Percentage enrichment of AT₂Rs with cellular subfractions through mitochondrial purification. Fractions: 1, whole-cell lysate; 2, postnuclear (480 × g); 3, postdifferential centrifugation (7,700 × g); 4, post-HistoDenz gradient centrifugation (50,500 × g); 5, postsucrose gradient centrifugation (77,000 × g).

Fig. 6.

mtAT₂R modulation of mitochondrial respiration and NO production. (A) Increased mitochondrial NO production in response to 10 nM and 100 nM concentrations of the AT₂R agonist CGP421140 (CGP), which can be reversed with the addition of a 1 μM concentration of the AT₂R antagonist PD-123319 (PD). (B) Mitochondrial respiration decreased significantly in response to serially increasing concentrations of CGP421140. Linear regression of CGP421140 concentrations versus oxygen consumption was significant at P < 0.0004. (C) Decreased respiration in response to AT₂R agonist CGP421140 at 100 nM was reversed with the addition of AT₂R antagonist PD-123319 at 1 μM or an inhibitor of NO production, l-NAME, at 100 nM. (D) No changes in mitochondrial membrane potential (ΔΨ_m) are evident in response to CGP421140 at increasing concentrations, confirming that effects observed on mitochondrial respiration are not attributable to nonspecific effects of CGP421140 on mitochondrial bioenergetic parameters. (*P < 0.05, **P < 0.005, ***P < 0.0005.)

Fig. 7.

Effect of AT₁R blocker losartan on expression of mitochondrial AT₁Rs and AT₂Rs. Renal tubular cell sections from C57BL/6 20 wks old (A and E), 70 wks old (B and F), and 70 wks old treated with losartan 40-60 mg/kg/day for 20 wks (C and G). D and H represent average counts of immune-labeling densities of mtAT₁R (D) and mtAT₂R (H) by age group in response to losartan. Gold particles in 30 mitochondria from each immunolabeling experiment were counted and averaged. Age was associated with a significant decrease in mtAT₂R that was reversed with chronic use of losartan. *P < 0.005, **P < 0.0005.