Central Ang II (Angiotensin II)-Mediated Sympathoexcitation

Figures

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  Figure 1.. Hemodynamic, neural and autonomic changes to Ang II. A, Mean arterial pressure (MAP) and heart rate (HR) measured by telemetry throughout the experiment in intracerebroventricular (ICV), saline (n=6), and Ang II (angiotensin II; n=7) infused rats. B, Representative digital images of FosB (Proto-Oncogene) staining in saline- and Ang II-infused rats. Each pair of saline- and Ang II-infused images were adjusted for uniform brightness and contrast; scale bar=100 μm, (C) the mean number of FosB-positive cells in the paraventricular nucleus of the hypothalamus. n=3–4/group. D, Cardiac sympathetic tone and (E) cardiac parasympathetic tone calculated by the changes in HR responses to methylatropine and propranolol, and (F) the ratio between these variables in the Saline (n=6) and Ang II (n=7) infused rats. T-test (with Welch correction; A, C, D, E, and F). **P=0.004, *P=0.048 (A); ***P=0.0007 (C); *P=0.021 (D); *P<0.042 (E); *P=0.042 (F) vs saline control.

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  Figure 2.. Changes in Baroreflex and chemoreflex evaluation after Ang II. A, Baroreflex sensitivity evaluated by tachycardiac and bradycardiac responses to changes in mean arterial pressure (MAP) induced by graded doses of sodium nitroprusside (0.5–5 µg, IV) or phenylephrine (0.25–2.5 µg, IE), respectively and expressed as the average of the ratio of the maximal changes in heart rate (HR) to a given change in MAP in saline (n=6) and Ang II (angiotensin II)-infused (n=8) rats. Statistical significance was assessed by t test (with Welch correction), **P=0.001,*P=0.014. B, Baroreflex sensitivity expressed as the best fit line that correlates change in HR vs change in MAP in Saline and Ang II-infused rats. Pearson correlation coefficient (r) were significant for tachycardia (saline, −0.74; Ang II, −0.54) and bradycardia (Ang II, −0.44) stages of the baroreflex. C, Changes in MAP and HR in response to potassium cyanide (KCN, 20–80 µg, IV) in saline and Ang II rats. Two-way ANOVA showed a significant effect of Ang II. *P=0.040 for ΔMAP, KCN 80 µg Ang II vs saline control (C).

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  Figure 3.. Changes in HIF-1α (hypoxia-inducible factor-1α) after Ang II. A, HIF-1α mRNA expression relative to rpl19 (ribosomal protein L 19) measured by real-time PCR in the paraventricular nucleus of the hypothalamus (PVN) and (B) cortex of vehicle and Ang II (angiotensin II)-infused group. The 2−ΔΔCT method was used to calculate relative changes in gene expression, which relates the expression of the target gene to the expression of a reference gene. Changes in HIF-1α mRNA are presented in folds with reference to the Saline group. C, Protein expression of HIF-1α expression in the PVN and (D) cortex of saline and Ang II-infused group top: a representative Western blot, bottom: densitometric analyses of HIF-1α level normalized to tubulin. E, Immunofluorescent staining of PVN sections from saline and Ang II-infused rats. HIF-1α (green) and nuclei (blue). F, Average values obtained by the reciprocal intensity method in each group were shown. Values are mean±SEM of analyses of 4 to 6 animals in each group. T-test (with Welch correction [A–D], and [F]). *P=0.010 (A); P=0.0721 (B); **P=0.004 (C); P=0.0626 (D); *P=0.016 (F) vs saline control.

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  Figure 4.. Changes in expression of NMDA-NR1 (N-methyl-D-aspartate NR1 receptor) after Ang II infusion. A, NMDA-NR1 mRNA expression relative to rpl19 (ribosomal protein L 19) measured by real-time polymerase chain reaction in the (A) paraventricular nucleus of the hypothalamus (PVN) and (B) cortex of saline and Ang II-infused group. Changes in NMDA-NR1 mRNA are presented in folds with reference to the Saline group. C, Protein expression of NMDA-NR1 expression in the PVN (C) and (D) cortex of saline and Ang II-infused group top: a representative Western blot, bottom: densitometric analyses of NMDA-NR1 level normalized to tubulin. Values are mean±SEM (n=4–6 rats per group). T-test with Welch correction (A–D). *P=0.037 (A); P=0.396 (B); *P=0.035 (C); P=0.189 (D) vs saline control.

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  Figure 5.. Reponses to NMDA (N-methyl-D-aspartate) in the PVN. Renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP), and heart rate (HR) responses to N-methyl-D-aspartate (NMDA) injected into the PVN. A, A segment of an original recording from an individual rat demonstrating the baseline parameters and peak changes in RSNA, integrated RSNA, MAP, and HR by the administration of 200 pmol of NMDA into the PVN in the 2 groups. B, Mean data of changes in RSNA, MAP, and HR following different doses of NMDA (50–200 pmol) of NMDA into the PVN of Saline and Ang II (angiotensin II) infused rats. Two-way ANOVA revealed significant interaction between effect of Ang II and dose of NMDA (P=0.031). *P=0.026 for ΔRSNA following 200 pmol of NMDA into the PVN vs saline control.

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  Figure 6.. Changes in expression of HIF-1α (hypoxia-inducible factor-1α) and NMDA-NR1 (N-methyl-D-aspartate NR1 receptor) after Ang II (angiotensin II) in NG108 cells. A, Western blot to assess levels of HIF-1α protein after treatment with different doses of Ang II. Top: a representative Western blot, bottom: densitometry analyses of HIF-1α level normalized to Actin, T-test (with Welch correction). **P=0.006 vs Control, ***P<0.0001 vs Control, *P=0.010 vs Control. B, Immunofluorescent staining for HIF-1α and NMDAR (N-methyl-D-aspartate [NMDA] receptor)-NMDA-type1 (NR1) after 8 h of Ang II treatment in NG108 cells. NMDA-NR1 (red), HIF-1α (green), and Nuclei (blue). C, Western blot of HIF-1α and NMDAR-NR1 expression after siRNA HIF-1α (8 pmol) knockdown (KD) and Ang II, 1 μM stimulation. D, Cumulative data of HIF-1α expression. Two-way ANOVA revealed significant interaction between effect of Ang II and HIF-1α KD (P=0.019). ***P=0.0001 Ang II vs Control, **P=0.008 HIF-1α KD vs Control, ***P=0.0001 Ang II+HIF-1α KD vs Ang II and (E) cumulative data of NMDA-NR1 expression. Two-way ANOVA revealed significant interaction between effect of Ang II and HIF-1α KD (P=0.032). **P=0.005 Ang II vs…