μ-Opioid Receptor-Mediated AT1R–TLR4 Crosstalk Promotes Microglial Activation to Modulate Blood Pressure Control in the Central Nervous System

Figures

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  Figure 1. Ang II supports superoxide generation, increases the activation of microglia and restores the nNOS pathway in the NTS of spontaneously hypertensive rats. (A,B) Graph showing systemic blood pressure (SBP) and nitric oxide (NO) concentrations in the nucleus tractus solitarius (NTS) of normotensive Wistar Kyoto rats (WKY; 6-, 20-week-old) and spontaneously hypertensive rats (SHRs; 6-, 20-week-old). The bar graph shows the NO concentration as μM nitrate per μg of total protein. (C) Representative images of DHE-treated brain sections, images photographed at ×280 magnification. ROS index in the NTS of the SHRs groups (20 weeks old) was compared to SHRs (6 weeks old). The ROS index is the relative mean fluorescence intensity for dihydroethidium. Sections including the NTS of SHRs rats displayed significant increase in DHE fluorescence compared with the SHRs (6 weeks old) group sections. (D) Quantitative immunoblotting analysis of nNOSS1416 phosphorylation in the NTS of 6-week-old WKY, SHRs and 20-week-old SHRs. One-way analysis of variance (ANOVA) with Scheffé post-hoc was performed for statistical analysis in Figure 1A–D. (E) Representative images for immunofluorescence staining of…

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  Figure 2. The downregulation of AT1R-induced superoxide generation is associated with the activation of microglia and the expression of TLR4 in the NTS of spontaneously hypertensive rats. (A) SBP after losartan administration for 2 weeks. (B) Representative images of DHE-treated brain sections, photographed at ×280 magnification. Bar graph representation of ROS index in the NTS of WKY, SHRs, and SHRs treated with losartan. (C,D) Representative fluorescence images for AT1R (green), TLR4 (red) and microglial marker IBA-1 (white)-positive cells in the NTS sections of WKY, SHRs, and SHRs treated with losartan. The images were photographed at ×400 and 1000 magnification. The Mann–Whitney U-test was performed for statistical analysis in Figure 1E. One-way analysis of variance (ANOVA) with Scheffé post-hoc was performed for statistical analysis. The values are presented as mean ± SEM. * p < 0.05 indicates significant difference from 20-week-old WKY. # p < 0.05 versus SHR. All data are presented as means ± SEM (n = 6 per group).

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  Figure 3. Activation of μOR in the NTS and cardiovascular regulation of WKY rats and SHRs. (A) Representative tracings demonstrating the cardiovascular effects of [D-Ala2, MePhe4, Gly5-ol]-enkephalin (DAMGO) (0.3 nmol) injected into the unilateral NTS of anesthetized WKY (▲ time of injection). (B) Representative BP recordings of an intra-NTS microinjection of the μOR antagonist CTAP in hypertensive SHRs (▲ time of injection). (C) Bar graphs showing the effects of 10-min treatment with DAMGO on the mean blood pressure (MBP) of anesthetized WKY. (D) Bar graphs showing the effects of 10-min treatment with DAMGO on the mean blood pressure (MBP) of anesthetized SHRs. (E,F) Representative red fluorescence images and the statistical analysis for Endomorphin-2-positive cells after DAMGO or CTAP treatment. The images were photographed at ×400 and 1000 magnification. The Mann–Whitney U-test was used for statistical analysis. Bar values are shown as mean ± SEM (n = 6); * p < 0.05 versus control.

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  Figure 4. μOR elevates the formation of AT1R and μOR heterodimers, and induces the activation of microglia and the expression of TLR4 to impair the nNOS pathway in the NTS. (A) The in situ PLA (Proximity Ligation Assay) was used to confirm the formation of AT1R and μOR (μ-opioid receptors) heterodimers after intra-NTS DAMGO or CTAP microinjection. Green color indicates AT1R and μOR heterodimers; the nuclei were counterstained with DAPI. The images were photographed at 1000 magnification. (B–E) Representative fluorescence images of AT1R (green), TLR4 (red) microglial marker IBA-1 (white) and nNOSS1416 (green)-positive cells after intra-NTS DAMGO or CTAP microinjection. The images were photographed at ×400 and 1000 magnification. The Mann–Whitney U-test was used for statistical analysis. Bar values are shown as mean ± SEM (n = 6); * p < 0.05 versus control.

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  Figure 5. Proposed pathogenic mechanism for neurogenic hypertension. (A) The interaction of Ang II and μOR enhances the binding of Ang II to the AT1R receptor, activates the microglia and promotes the formation of AT1R-μOR heterodimers in the NTS, leading to superoxide production. This inactivates nNOS-derived NO, causing systemic elevations in blood pressure. (B) AT1R inhibitors (such as losartan) decrease superoxide production, abolish the activation of microglia and AT1R, and decrease TLR4 expression, which ultimately leads to improved hypertension (red line). Interestingly, μOR inhibitors (such as CTAP) decrease BP and abolish μOR-induced formation of AT1R and μOR heterodimers. CTAP also significantly inactivates the activation of microglia and AT1R, and reduces TLR4 expression, which can lead to an increase nNOS-derived NO levels, thereby improving hypertension (black line). In summary, this study shows how the interaction between Ang II and μOR enhances the binding of Ang II to AT1R, thereby causing microglia activation and inducing superoxide production, which, in turn, leads to neurotoxicity. Furthermore, the interaction of Ang II and μOR also enhances the formation of the AT1R and…