Brain nuclear factor-kappa B activation contributes to neurohumoral excitation in angiotensin II-induced hypertension

Abstract

Aims: Angiotensin II (ANG II)-induced inflammatory and oxidative stress responses contribute to the pathogenesis of hypertension. In this study, we determined whether nuclear factor-kappa B (NF-kappaB) activation in the hypothalamic paraventricular nucleus (PVN) increases oxidative stress and contributes to the ANG II-induced hypertensive response.

Methods and results: Rats were infused intravenously with ANG II (10 ng/kg per min) or saline for 4 weeks. These rats received either vehicle or losartan (LOS, 20 microg/h), an angiotensin II type 1 receptor (AT1-R) antagonist; pyrrolidine dithiocarbamate (PDTC, 5 microg/h), a NF-kappaB inhibitor; tempol (TEMP, 80 microg/h), a superoxide scavenger; LOS (20 microg/h), and PDTC (5 microg/h); or TEMP (80 microg/h) and PDTC (5 microg/h), given intracerebroventricularly (ICV) via osmotic minipump. ANG II infusion resulted in increased mean arterial pressure, renal sympathetic nerve activity, plasma proinflammatory cytokines (PIC), norepinephrine, and aldosterone. These rats also had higher levels of Fra-LI (an indicator of chronic neuronal activation), PIC, phosphorylated IKKbeta, NF-kappaB subunits, AT1-R, superoxide, and gp91phox (a subunit of NADP(H) oxidase) and lower levels of IkappaBalpha in the PVN than control animals. ICV treatment with LOS, PDTC, or TEMP attenuated these changes, and combined treatment with ICV LOS and PDTC, or ICV TEMP and PDTC prevented these ANG II-induced hypertensive responses.

Conclusion: These findings suggest that an ANG II-induced increase in the brain renin-angiotensin system activates NF-kappaB in the PVN and contributes to sympathoexcitation in hypertension. The increased superoxide in the PVN contributes to NF-kappaB activation and neurohumoral excitation in hypertension.

Figure 1

Effect of intracerebroventricular (ICV) treatment with artificial cerebrospinal fluid (aCSF), losartan (LOS), pyrrolidine dithiocarbamate (PDTC), tempol (TEMP), LOS + PDTC, or TEMP + PDTC on mean arterial pressure (MAP) and angiotensin II type 1 receptor (AT1-R) in the paraventricular nucleus (PVN) of angiotensin II (ANG II)-infused rats and control rats. (A) Mean arterial pressure in different groups. (B) Angiotensin II type 1 receptor mRNA expression in different groups. (C) Western blot of angiotensin II type 1 receptor in the paraventricular nucleus in different groups. *P < 0.05 vs. control (NS + Treated or NS + ICV aCSF); ^†P < 0.05 ANG II + Treated vs. ANG II + ICV aCSF; ^#P < 0.05 ANG II + ICV PDTC vs. ANG II + ICV aCSF; ^ΔP < 0.05 ANG II + ICV LOS vs. ANG II + ICV aCSF; ^$P < 0.05 ANG II + ICV TEMP vs. ANG II + ICV aCSF; ^‡P < 0.05 ANG II + ICV LOS + PDTC vs. ANG II + ICV aCSF; ^&P < 0.05 ANG II + ICV TEMP + PDTC vs. ANG II + ICV aCSF.

Figure 2

Effect of intracerebroventricular (ICV) treatment with artificial cerebrospinal fluid (aCSF), losartan (LOS), pyrrolidine dithiocarbamate (PDTC), tempol (TEMP), LOS + PDTC, or TEMP + PDTC on renal sympathetic nerve activity (RSNA: % max) in angiotensin II (ANG II)-infused rats and control rats. (A) Renal sympathetic nerve activity in different groups. (B) Bar graph comparing renal sympathetic nerve activity in different groups. *P < 0.05 vs. control (NS + Treated or NS + ICV aCSF). ^†P < 0.05 ANG II + Treated vs. ANG II + ICV aCSF.

Figure 3

Effect of intracerebroventricular (ICV) treatment with artificial cerebrospinal fluid (aCSF), losartan (LOS), pyrrolidine dithiocarbamate (PDTC), tempol (TEMP), LOS + PDTC, or TEMP + PDTC on Fra-like (Fra-LI), tumour necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) expression in the paraventricular nucleus (PVN) of angiotensin II (ANG II)-infused rats and control rats. (A) Immunohistochemistry for Fra-like (black dots), tumour necrosis factor-alpha (blue), interleukin-1β (pink), and interleukin-6 (pink) positive neurons in different groups. (B) Bar graph comparing Fra-like, tumour necrosis factor-alpha, interleukin-1β, and interleukin-6 positive neurons in different groups. *P < 0.05 vs. control (NS + Treated or NS + ICV aCSF). ^†P < 0.05 ANG II + Treated vs. ANG II + ICV aCSF.

Figure 4

Effect of intracerebroventricular (ICV) treatment with artificial cerebrospinal fluid (aCSF), losartan (LOS), pyrrolidine dithiocarbamate (PDTC), tempol (TEMP), LOS + PDTC, or TEMP + PDTC on phosphorylated IKKβ (p-IKKβ) and NF-κB p50 in the paraventricular nucleus (PVN) of angiotensin II (ANG II)-infused rats and control rats. (A) Immunofluorescence for phosphorylated IKKβ (bright red) and NF-κB p50 (bright green) positive neurons in different groups. Neuronal nuclei are shown in blue. (B) Bar graph comparing paraventricular nucleus phosphorylated IKKβ and NF-κB p50 positive neurons in different groups. *P < 0.05 vs. control (NS + Treated or NS + ICV aCSF). ^†P < 0.05 ANG II + Treated vs. ANG II + ICV aCSF.

Figure 5

Effect of intracerebroventricular (ICV) treatment with artificial cerebrospinal fluid (aCSF), losartan (LOS), pyrrolidine dithiocarbamate (PDTC), tempol (TEMP), LOS + PDTC, or TEMP + PDTC on mRNA expression for NF-κB p65 and IκBα, and protein expression for phosphorylated IKKβ (p-IKKβ) and gp91^phox in the paraventricular nucleus (PVN) of angiotensin II (ANG II)-infused rats and control rats. (A) NF-κB p65 mRNA expression in the paraventricular nucleus in different groups. (B) IκBα mRNA expression in the paraventricular nucleus in different groups. (C) Western blot of phosphorylated IKKβ in the paraventricular nucleus in different groups. (D) Western blot of the NADP(H) oxidase subunit gp91^phox in the paraventricular nucleus in different groups. *P < 0.05 vs. control (NS + Treated or NS + ICV aCSF). ^†P < 0.05 ANG II + Treated vs. ANG II + ICV aCSF.

Figure 6

Effect of intracerebroventricular (ICV) treatment with artificial cerebrospinal fluid (aCSF), losartan (LOS), pyrrolidine dithiocarbamate (PDTC), tempol (TEMP), LOS + PDTC, or TEMP + PDTC on superoxide and NADP(H) oxidase in the paraventricular nucleus (PVN) of angiotensin II (ANG II)-infused rats and control rats. (A) Immunofluorescence for the NADP(H) oxidase subunit gp91^phox (bright green) and superoxide as determined by fluorescent-labelled dihydroethidium (DHE, bright red) in the paraventricular nucleus in different groups. (B) Immunofluorescent intensity of gp91^phox and dihydroethidium in the paraventricular nucleus of different groups of rats. (C) Comparison of gp91^phox positive neurons in the paraventricular nucleus in different groups. *P < 0.05 vs. control (NS + Treated or NS + ICV aCSF). ^†P < 0.05 ANG II + Treated vs. ANG II + ICV aCSF.

Figure 7

The schematic of the hypothesis showing the mechanism by which angiotensin II modulates nuclear factor-kappa B activation in the paraventricular nucleus and contributes to neurohumoral excitation and pressor response in hypertension.