Aging enhances pressure-induced arterial superoxide formation

Abstract

The purpose of this study was to investigate the mechanisms that regulate superoxide (O(2)(*-)) production as a function of an acute elevation of intravascular pressure and age. Mesenteric arteries isolated from young (6 mo) and aged (24 mo) male Fischer 344 rats were used. O(2)(*-) production in vessels in response to 80 (normal pressure, NP) and 180 (high pressure, HP) mmHg was determined by the superoxide dismutase-inhibitable nitroblue tetrazolium (NBT) reduction assay. In vessels exposed to NP, O(2)(*-) production was significantly higher in aged than in young vessels (32.7 +/- 7.0 vs. 15.4 +/- 2.4 nmol.mg(-1).30 min(-1)). HP enhanced O(2)(*-) production in vessels of both groups, but the enhancement was significantly greater in aged than in young vessels (63.4 +/- 6.7 vs. 32.7 +/- 4.3 nmol.mg(-1).30 min(-1)). Apocynin (100 micromol/l) attenuated HP-induced increases in O(2)(*-) production in both groups, whereas allopurinol (100 micromol/l) and N(omega)-nitro-L-arginine methyl ester (100 mumol/l) inhibited the response only in aged vessels. Confocal microscopy showed increases in O(2)(*-) in response to HP in endothelial and smooth muscle layers of both groups, with much greater fluorescent staining in aged than in young rats and in the endothelium than in smooth muscle cells. No significant changes in NAD(P)H oxidase gene and protein expressions were observed in vessels of the two groups. Upregulation of protein expression of xanthine oxidase was detected in aged vessels. We conclude that NAD(P)H oxidase contributes importantly to HP-induced enhanced O(2)(*-) production in vessels of both young and aged rats, whereas xanthine oxidase and nitric oxide synthase-dependent O(2)(*-) production also contribute to the enhancement in mesenteric arteries of aged rats.

Fig. 1

Nitroblue tetrazolium (NBT) reduction assay detecting ${\mathrm{O}}_2^{•-}$ production in mesenteric arteries of 6- and 24-mo-old male Fischer rats in response to 0, 80, and 180 mmHg of intravascular pressure. *Significant difference vs. 6-mo-old rats. ≠ and #Significant differences vs. 0 and 80 mmHg, respectively.

Fig. 2

${\mathrm{O}}_2^{•-}$ production in response to 180 mmHg of intravascular pressure in control (Ctr) and after inhibition of NADPH oxidase with apocynin (Apo, 10⁻⁴ M), xanthine oxidase with allopurinol (Allop, 10⁻⁴ M), and nitric oxide synthase with N^ω-nitro-l-arginine methyl ester (l-NAME, 10⁻⁴ M), respectively, in mesenteric arteries of 6- (A) and 24-mo-old rats (B). *Significant difference vs. control. n, Number of rats.

Fig. 3

A and B: representative Western blot analyses of NADPH oxidase subunits Nox-1, gp91phox, and Nox-4, and xanthine oxidase (XO), respectively, in mesenteric arteries of 6- and 24-mo-old rats. β-Actin was used to normalize for loading variations. C and D: summarized data of three Western blot analyses (12 rats in each group). Data are normalized by means of densitometric ratios of proteins of interest and β-actin from vessels of 6-mo-old rats. *Significant difference vs. 6-mo-old rats.

Fig. 4

Expression of gp91phox (A) and Nox-4 (B) mRNA in mesenteric vessels exposed to 80 and 180 mmHg for 10, 30, and 120 min, of 6- and 24-mo-old rats. Controls were freshly isolated vessels without pressure treatment. Quantitative RT-PCR was used to determine expression of gp91-phox and Nox-4 relative to β-actin expression as described in MATERIALS AND METHODS.

Fig. 5

A: sample images of confocal microscopy comparing fluorescence intensity of dihydroxyethidium (DHE) (red color) in response to 80 and 180 mmHg of intravascular pressure in the endothelial and smooth muscle layers of mesenteric arteries of 6-mo-old rats. a and b are the endothelial layers; c and d are smooth muscle layers; a and c, and b and d are vessels treated with 80 and 180 mmHg of pressure, respectively. B: summarized histogram data detecting fluorescence intensity of DHE in the endothelial and smooth muscle layers of 6- and 24-mo-old rats. *Significant difference compared with 80 mmHg. #Significant difference compared with 6-mo-old rats. C: percent increase in fluorescence intensity from 80 to 180 mmHg of intravascular pressure. *Significant difference compared with 6-mo-old rats. #Significant difference compared with EC.