Nitro-oleic acid inhibits angiotensin II-induced hypertension

Abstract

Rationale: Nitro-oleic acid (OA-NO(2)) is a bioactive, nitric-oxide derived fatty acid with physiologically relevant vasculoprotective properties in vivo. OA-NO(2) exerts cell signaling actions as a result of its strong electrophilic nature and mediates pleiotropic cell responses in the vasculature.

Objective: The present study sought to investigate the protective role of OA-NO(2) in angiotensin (Ang) II-induced hypertension.

Methods and results: We show that systemic administration of OA-NO(2) results in a sustained reduction of Ang II-induced hypertension in mice and exerts a significant blood pressure lowering effect on preexisting hypertension established by Ang II infusion. OA-NO(2) significantly inhibits Ang II contractile response as compared to oleic acid (OA) in mesenteric vessels. The improved vasoconstriction is specific for the Ang II type 1 receptor (AT(1)R)-mediated signaling because vascular contraction by other G-protein-coupled receptors is not altered in response to OA-NO(2) treatment. From the mechanistic viewpoint, OA-NO(2) lowers Ang II-induced hypertension independently of peroxisome proliferation-activated receptor (PPAR)gamma activation. Rather, OA-NO(2), but not OA, specifically binds to the AT(1)R, reduces heterotrimeric G-protein coupling, and inhibits IP(3) (inositol-1,4,5-trisphosphate) and calcium mobilization, without inhibiting Ang II binding to the receptor.

Conclusions: These results demonstrate that OA-NO(2) diminishes the pressor response to Ang II and inhibits AT(1)R-dependent vasoconstriction, revealing OA-NO(2) as a novel antagonist of Ang II-induced hypertension.

Figure 1. OA-NO₂ inhibits Ang II-induced hypertension

Mice implanted with osmotic pumps adjusted to deliver 5 mg/kg/day OA-NO₂ (red) or OA (blue) and vehicle control (black) were infused with Ang II at a pressor rate of 500 ng/kg/min. Systolic (A) and diastolic (C) blood pressure was measured by radiotelemetry. Data are shown as mean ± SEM, n=6 in each group, *P < 0.05. OA-NO₂-treated mice showed significantly lower systolic (~15 mmHg reduction) (B) and diastolic pressure (~12 mmHg reduction) (D) after Ang II challenge than did OA and vehicle-treated mice as determined both at the night and day time periods. Data in B and D are shown as mean ± SEM of measurements collected before (3 days) and after Ang II (12 days), n=6 in each group, *P < 0.05.

Figure 1. OA-NO₂ inhibits Ang II-induced hypertension

Mice implanted with osmotic pumps adjusted to deliver 5 mg/kg/day OA-NO₂ (red) or OA (blue) and vehicle control (black) were infused with Ang II at a pressor rate of 500 ng/kg/min. Systolic (A) and diastolic (C) blood pressure was measured by radiotelemetry. Data are shown as mean ± SEM, n=6 in each group, *P < 0.05. OA-NO₂-treated mice showed significantly lower systolic (~15 mmHg reduction) (B) and diastolic pressure (~12 mmHg reduction) (D) after Ang II challenge than did OA and vehicle-treated mice as determined both at the night and day time periods. Data in B and D are shown as mean ± SEM of measurements collected before (3 days) and after Ang II (12 days), n=6 in each group, *P < 0.05.

Figure 1. OA-NO₂ inhibits Ang II-induced hypertension

Mice implanted with osmotic pumps adjusted to deliver 5 mg/kg/day OA-NO₂ (red) or OA (blue) and vehicle control (black) were infused with Ang II at a pressor rate of 500 ng/kg/min. Systolic (A) and diastolic (C) blood pressure was measured by radiotelemetry. Data are shown as mean ± SEM, n=6 in each group, *P < 0.05. OA-NO₂-treated mice showed significantly lower systolic (~15 mmHg reduction) (B) and diastolic pressure (~12 mmHg reduction) (D) after Ang II challenge than did OA and vehicle-treated mice as determined both at the night and day time periods. Data in B and D are shown as mean ± SEM of measurements collected before (3 days) and after Ang II (12 days), n=6 in each group, *P < 0.05.

Figure 1. OA-NO₂ inhibits Ang II-induced hypertension

Mice implanted with osmotic pumps adjusted to deliver 5 mg/kg/day OA-NO₂ (red) or OA (blue) and vehicle control (black) were infused with Ang II at a pressor rate of 500 ng/kg/min. Systolic (A) and diastolic (C) blood pressure was measured by radiotelemetry. Data are shown as mean ± SEM, n=6 in each group, *P < 0.05. OA-NO₂-treated mice showed significantly lower systolic (~15 mmHg reduction) (B) and diastolic pressure (~12 mmHg reduction) (D) after Ang II challenge than did OA and vehicle-treated mice as determined both at the night and day time periods. Data in B and D are shown as mean ± SEM of measurements collected before (3 days) and after Ang II (12 days), n=6 in each group, *P < 0.05.

Figure 2. OA-NO₂ inhibits the pressor response to Ang II

BP was monitored upon treatment with OA (A) and OA-NO₂ (B) (10 mg/kg) before Ang II infusion (10μg/mL, infusion rate: 1μL/min). All drugs were delivered via jugular vein administration starting at the time indicated by the arrows. BP tracings were recorded by insertion of a micro-tip catheter sensor into the right carotid artery. (C) Systolic BP was averaged for a 10 min period before and after Ang II infusion (10μg/mL, infusion rate: 1μL/min). Data are shown as mean ± SEM, n=6 in each group. OA-NO₂ significantly reduced the pressor response to Ang II infusion compared to OA. P < 0.05 (OA-NO₂ vs. OA after Ang II infusion). (C) Hypertension was developed in mice in 3 days after implantation of osmotic pumps to infuse Ang II at a pressor rate of 500 ng/kg/min. OA (D) or OA-NO₂ (E) were then delivered to mice via the jugular vein at increasing concentrations as indicated by the arrows. BP tracings were recorded as described above. (C) Data in D and E are summarized as systolic BP reduction (in mmHg) after either OA or OA-NO₂ treatment as compared to maximal systolic pressure in Ang II hypertensive mice. OA-NO₂ treatment but not OA dose-dependently reduced established hypertension upon Ang II delivery. Data are shown as mean ± SEM, n=4 in each group, *P < 0.05.

Figure 2. OA-NO₂ inhibits the pressor response to Ang II

BP was monitored upon treatment with OA (A) and OA-NO₂ (B) (10 mg/kg) before Ang II infusion (10μg/mL, infusion rate: 1μL/min). All drugs were delivered via jugular vein administration starting at the time indicated by the arrows. BP tracings were recorded by insertion of a micro-tip catheter sensor into the right carotid artery. (C) Systolic BP was averaged for a 10 min period before and after Ang II infusion (10μg/mL, infusion rate: 1μL/min). Data are shown as mean ± SEM, n=6 in each group. OA-NO₂ significantly reduced the pressor response to Ang II infusion compared to OA. P < 0.05 (OA-NO₂ vs. OA after Ang II infusion). (C) Hypertension was developed in mice in 3 days after implantation of osmotic pumps to infuse Ang II at a pressor rate of 500 ng/kg/min. OA (D) or OA-NO₂ (E) were then delivered to mice via the jugular vein at increasing concentrations as indicated by the arrows. BP tracings were recorded as described above. (C) Data in D and E are summarized as systolic BP reduction (in mmHg) after either OA or OA-NO₂ treatment as compared to maximal systolic pressure in Ang II hypertensive mice. OA-NO₂ treatment but not OA dose-dependently reduced established hypertension upon Ang II delivery. Data are shown as mean ± SEM, n=4 in each group, *P < 0.05.

Figure 3. OA-NO₂ reduces AT₁R-dependent vessel contraction

Second-grade mesenteric artery rings were mounted into a myograph as described in the Online Material. The mesenteric artery rings were preincubated for 10 min with 2.5 μmol/L or 5 μmol/L of either OA or OA-NO₂ as indicated. Vascular contraction was induced by serial addition of increasing concentrations of Ang II (A), PE (B) or ET-1 (C) to the myograph. Contractile response was quantified and shown in the figure as % of the vessel contraction force afforded by 50 mmol/L KCl. OA-NO₂ dose-dependently reduced Ang II induced vasoconstriction but not that of PE or ET-1. Data are shown as mean ± SEM, n=6 in each group, *P < 0.05 and **P < 0.01 (OA-NO₂ vs. OA or vehicle (0.1% ethanol-treated arteries).

Figure 4. OA-NO₂ reduces Ang II-induced hypertension independently of PPARγ activation

BP in mice was monitored with a micro-tip catheter sensor inserted into the right carotid artery by arteriotomy upon treatment with OA (A) and OA-NO₂ (B) (10 mg/kg) delivered via jugular vein administration after infusion of the PPARγ inhibitor, GW9662 (10 mg/kg). (A) and (B) show representative BP recordings of n=6 in each treatment before and after Ang II infusion (10μg/mL), delivered at an infusion rate of 1μL/min. (C) OA-NO₂ delivery results in a significant reduction of Ang II-induced hypertension (~10 mmHg) independently of PPARγ inhibition by GW9662. Data are depicted as systolic BP increase (in mmHg) after Ang II infusion as compared to baseline systolic pressure and are shown as mean ± SEM, n=6 in each group, P < 0.01 (OA-NO₂ vs. OA in either GW9662-treated or DMSO vehicle control-treated group after Ang II infusion). (D) The mesenteric artery rings were preincubated for 10 min with 10 μmol/L of GW9662 and then with 2.5 μmol/L of either OA or OA-NO₂ as indicated. Vascular contraction was induced by serial addition of increasing concentrations of Ang II to the bath in the myograph. Contractile response was quantified and shown in the figure as % of the vessel contraction force afforded by 50 mmol/L KCl. OA-NO₂ reduced Ang II induced vasoconstriction independent of the PPARγ antagonist, GW9662. Data are shown as mean ± SEM, n=6 in each group, *P < 0.05.

Figure 5. OA-NO₂ forms covalent adducts with AT₁R but does not affect Ang II binding to the receptor

(A) HEK293 cells were transfected with 500 ng FLAG-tagged AT₁R plasmid (pcDNA3.1-AT₁R) versus control plasmid (pcDNA3.1) and treated with OA or OA-NO₂ as indicated. Immunoprecipitated AT₁R was subjected to a BME-based trans-nitroalkylation reaction as detailed in the Online Material. Adducted OA-NO₂ to AT₁R was released as BME-OA-NO₂ and quantified using mass spectrometry. The chromatographic profile shows a dose-dependent increase of OA-NO₂ after immunoprecipitation against AT₁R and verified by coelution with [¹³C₁₈]-OA-NO₂ internal standard (lower panel). (B) The fragmentation pattern (upper panel) of AT₁R-derived OA-NO₂ (5 μmol/L treatment) adducted to BME was confirmed by comparison with that of [¹³C₁₈]-OA-NO₂ (lower panel). Enhanced levels of the ion products in the peaks and comparison to the internal standard confirmed the structure for the proposed adducts formed after trans-nitroalkylation. The data shown is a representative set of 3 independent experimental repetitions. (C) Quantification of the AT₁R-derived BME adducts after nucleophilic exchange of OA-NO₂ from the AT₁R to BME, reveal a significant increase of adducted OA-NO₂ to the AT₁R (270 pmol/L vs. 13 pmol/L BME-OA-NO₂, respectively after 5 μmol/L OA-NO₂ treatment). (D) HEK293 cells were similarly transfected with the pcDNA3.1-AT₁R plasmid and treated with biotin-labeled OA-NO₂ or OA for 3 h at the indicated concentrations. Biotin-labeled adducts were visualized by Western blot after immunoprecipitation of the Flag-AT₁R. OA-NO₂ dose-dependently increased the covalent adduction to the immunoprecipitated AT₁R. The data shown is a representative set of 3 independent experimental repetitions. (E) Competition binding assays were performed using confluent VSMC pretreated with 2.5μmol/L of OA or OA-NO₂ for 10 min and then incubated for 30 min with 0.3 nmol/L ¹²⁵I-[Sar¹,Ile⁸]-Ang II and different concentrations of non-radiolabeled Ang II as indicated. Data are shown as % of maximal binding with ¹²⁵I-[Sar¹,Ile⁸]-Ang II. (F) Similarly, VSMC treated with different doses of OA or OA-NO₂ as indicated were then incubated for 30 min at 37°C with 0.1 nmol/L of ¹²⁵I-[Sar¹,Ile⁸]-Ang II. The ARB losartan (1μmol/L) served as a positive control. In panels E and F, radioactivity was measured using a γ-counter and values are expressed as mean ± SEM (n=4). The experiments were performed three times with similar results.

Figure 5. OA-NO₂ forms covalent adducts with AT₁R but does not affect Ang II binding to the receptor

(A) HEK293 cells were transfected with 500 ng FLAG-tagged AT₁R plasmid (pcDNA3.1-AT₁R) versus control plasmid (pcDNA3.1) and treated with OA or OA-NO₂ as indicated. Immunoprecipitated AT₁R was subjected to a BME-based trans-nitroalkylation reaction as detailed in the Online Material. Adducted OA-NO₂ to AT₁R was released as BME-OA-NO₂ and quantified using mass spectrometry. The chromatographic profile shows a dose-dependent increase of OA-NO₂ after immunoprecipitation against AT₁R and verified by coelution with [¹³C₁₈]-OA-NO₂ internal standard (lower panel). (B) The fragmentation pattern (upper panel) of AT₁R-derived OA-NO₂ (5 μmol/L treatment) adducted to BME was confirmed by comparison with that of [¹³C₁₈]-OA-NO₂ (lower panel). Enhanced levels of the ion products in the peaks and comparison to the internal standard confirmed the structure for the proposed adducts formed after trans-nitroalkylation. The data shown is a representative set of 3 independent experimental repetitions. (C) Quantification of the AT₁R-derived BME adducts after nucleophilic exchange of OA-NO₂ from the AT₁R to BME, reveal a significant increase of adducted OA-NO₂ to the AT₁R (270 pmol/L vs. 13 pmol/L BME-OA-NO₂, respectively after 5 μmol/L OA-NO₂ treatment). (D) HEK293 cells were similarly transfected with the pcDNA3.1-AT₁R plasmid and treated with biotin-labeled OA-NO₂ or OA for 3 h at the indicated concentrations. Biotin-labeled adducts were visualized by Western blot after immunoprecipitation of the Flag-AT₁R. OA-NO₂ dose-dependently increased the covalent adduction to the immunoprecipitated AT₁R. The data shown is a representative set of 3 independent experimental repetitions. (E) Competition binding assays were performed using confluent VSMC pretreated with 2.5μmol/L of OA or OA-NO₂ for 10 min and then incubated for 30 min with 0.3 nmol/L ¹²⁵I-[Sar¹,Ile⁸]-Ang II and different concentrations of non-radiolabeled Ang II as indicated. Data are shown as % of maximal binding with ¹²⁵I-[Sar¹,Ile⁸]-Ang II. (F) Similarly, VSMC treated with different doses of OA or OA-NO₂ as indicated were then incubated for 30 min at 37°C with 0.1 nmol/L of ¹²⁵I-[Sar¹,Ile⁸]-Ang II. The ARB losartan (1μmol/L) served as a positive control. In panels E and F, radioactivity was measured using a γ-counter and values are expressed as mean ± SEM (n=4). The experiments were performed three times with similar results.

Figure 6. OA-NO₂ uncouples Gα_q11 to the AT₁R and inhibits Ang II-induced IP₃ production and Ca²⁺ mobilization in VSMC

(A) HEK293 cells cultured in 6-well plates were cotransfected with HA-tagged AT₁R (0.3 μg), Gα_q11-EGFP (0.3 μg), Gβ₁ (0.1 μg) and Gγ₂ (0.1 μg) plasmids and treated with either OA, OA-NO₂ (2.5 μmol/L) or losartan (1μmol/L) 30min before stimulation with Ang II (100 nmol/L) for 1 min. Immunoprecipitated Gα_q11 using a GFP antibody was subjected to deglycosylation before Western blot analysis for detection of HA-tagged AT₁R. OA-NO₂ but not OA reduced AT₁R coupled to the immunoprecipitated Gα_q11.The data shown is representative of 3 independent experiments. (B) Western blots were quantified using the ImageJ software after densitometric scanning of the films and expressed as relative ratio of AT₁R vs. Gα_q11. n=3. P < 0.05 OA-NO₂ vs. OA in Ang II-treated cells and P < 0.05 Ang II-treated vs. vehicle control cells (Con). (C) VSMC were treated with either 2.5 μmol/L OA-NO₂ or OA and then pulsed with 100 nmol/L Ang II for 45 min. IP₃ production was monitored by radioreceptor assays. Ang II induced a 2-fold increase of intracellular IP₃, whereas OA-NO₂, but not OA blocked Ang II-mediated IP₃ production, n=6. P < 0.05 OA-NO₂ vs. OA in Ang II-treated cells and P < 0.05 Ang II-treated vs. vehicle control cells (Con). (B) VSMC were similarly treated with either OA-NO₂ or OA (2.5μmol/L) and then pulsed with Ang II (100 nmol/L) in the presence of fura-2. Control experiments were performed with losartan (1 μmol/L) pretreated cells. Emission ratio between 340 nm and 380 nm was used to calculate intracellular calcium concentration as described in Online Material. The data shown depicts a representative time tracing of 6 independent experiments. The ratio-time traces were generated by averaging three typical cells in the field of view as described in Online Figure VI and Online Movies I through III).