Enhanced angiotensin II-mediated central sympathoexcitation in streptozotocin-induced diabetes: role of superoxide anion

Abstract

Studies have shown that the superoxide mechanism is involved in angiotensin II (ANG II) signaling in the central nervous system. We hypothesized that ANG II activates sympathetic outflow by stimulation of superoxide anion in the paraventricular nucleus (PVN) of streptozotocin (STZ)-induced diabetic rats. In α-chloralose- and urethane-anesthetized rats, microinjection of ANG II into the PVN (50, 100, and 200 pmol) produced dose-dependent increases in renal sympathetic nerve activity (RSNA), arterial pressure (AP), and heart rate (HR) in control and STZ-induced diabetic rats. There was a potentiation of the increase in RSNA (35.0 ± 5.0 vs. 23.0 ± 4.3%, P < 0.05), AP, and HR due to ANG II type I (AT(1)) receptor activation in diabetic rats compared with control rats. Blocking endogenous AT(1) receptors within the PVN with AT(1) receptor antagonist losartan produced significantly greater decreases in RSNA, AP, and HR in diabetic rats compared with control rats. Concomitantly, there were significant increases in mRNA and protein expression of AT(1) receptor with increased superoxide levels and expression of NAD(P)H oxidase subunits p22(phox), p47(phox), and p67(phox) in the PVN of rats with diabetes. Pretreatment with losartan (10 mg·kg(-1)·day(-1) in drinking water for 3 wk) significantly reduced protein expression of NAD(P)H oxidase subunits (p22(phox) and p47(phox)) in the PVN of diabetic rats. Pretreatment with adenoviral vector-mediated overexpression of human cytoplasmic superoxide dismutase (AdCuZnSOD) within the PVN attenuated the increased central responses to ANG II in diabetes (RSNA: 20.4 ± 0.7 vs. 27.7 ± 2.1%, n = 6, P < 0.05). These data support the concept that superoxide anion contributes to an enhanced ANG II-mediated signaling in the PVN involved with the exaggerated sympathoexcitation in diabetes.

Fig. 1.

A, a–c: schematic representations of serial sections from the rostral (−1.4) to the caudal (−2.1) extent of the region of the paraventricular nucleus (PVN). The distance (in mm) posterior to bregma is shown for each section. Each filled circle represents the site of termination of an injection that is considered to be within the PVN region in the control group; a plus symbol (+) represents the same in the diabetic group. d: Histological photo showing the injection site (arrow) in the PVN of one rat. AH, anterior hypothalamic nucleus; f, fornix; 3V, third ventricle; OX, optic tract; SO, supraoptic nucleus. Bars, 0.5 mm. B: segments of original recordings from an individual control rat (top) and diabetic rat (bottom) demonstrating the representative response to heart rate (HR), arterial pressure (AP), integrated renal sympathetic nerve activity (Int. RSNA), and RSNA to the microinjections of ANG II into the PVN. C: mean changes (Δ) in HR, mean arterial pressure (MAP), and RSNA after microinjections of ANG II into the PVN (50–200 pmol) and intravenous injection (500 pmol iv) in control and diabetic rats. *P < 0.05 vs. control group. bpm, Beats/min.

Fig. 2.

Mean changes in RSNA, mean arterial pressure (MAP), and HR after microinjections of losartan (25–100 nmol) into the PVN in control and diabetic rats. *P < 0.05 vs. control group.

Fig. 3.

A: gene expression of ANG II type I (AT₁) receptors in the PVN tissue measured. Mean band densities of AT₁ receptors were normalized to that of the reference gene, ribosomal protein L19 (rpl19), in control and diabetic rats. B: protein level of AT₁ receptors in the PVN tissue. Top, example of visualized bands of AT₁ receptors and β-tubulin; bottom, mean band densities of AT₁ receptors normalized to β-tubulin. *P < 0.05 vs. control group.

Fig. 4.

A: immunofluorescence photomicrographs from sections of the PVN region stained for dihydroethidium (DHE; red). Bar, 0.5 mm. B: quantification of DHE intensity in the PVN. *P < 0.05 vs. control group.

Fig. 5.

A: gene expression of p22^phox, p47^phox, and p67^phox in the PVN tissue measured. Mean p22^phox, p47^phox, and p67^phox mRNA expression were normalized to expression of rpl19. B: protein levels of p22^phox, p47^phox, and p67^phox in the PVN tissue. Top, example of visualized bands of p22^phox, p47^phox, p67^phox, and β-tubulin; bottom, mean band densities normalized to β-tubulin. *P < 0.05 vs. control group.

Fig. 6.

A: example of visualized bands of p22^phox, p47^phox, and β-tubulin in the PVN in losartan treatment experiment. Los, losartan. B: mean band densities normalized to β-tubulin. *P < 0.05 vs. control group. #P < 0.05 vs. diabetic group. C: immunofluorescence photomicrographs from sections of the PVN region stained for DHE (red). Bar, 0.5 mm. D: quantification of DHE intensity in the PVN.

Fig. 7.

A: immunofluorescence photomicrographs from sections of the PVN region stained for CuZn superoxide dismutase (CuZnSOD; red) in a diabetic rat. Arrow points to the site infected with adenoviral vector carrying CuZnSOD (AdCuZnSOD). Bar, 0.5 mm. B: real-time PCR quantification of CuZnSOD in the PVN after AdCuZnSOD transfection. C: segments of original recordings from individual control + adenoviral vector carrying the bacterial β-galactosidase gene (AdβGal), diabetic + AdβGal, control + AdCuZnSOD, and diabetic + AdCuZnSOD transfected rats, demonstrating the representative responses to HR, AP, int. RSNA, and RSNA to the microinjections of ANG II into the PVN. D: mean changes in RSNA, MAP, and HR after microinjections of ANG II into the PVN in 4 groups of rats. *P < 0.05 vs. control group. #P < 0.05 vs. diabetic + AdβGal group.