Recently we have demonstrated that in the heart that the acidosis that develops during ischaemia, is sufficient to chemically reduce nitrite back to NO, and that this is a process dependent on xanthine oxidase (XO) activity [1]. This mechanism provides an alternative source of protective NO during ischaemia where conventional NO synthesis by NO synthase has been compromised. However whether the activity resided in the myocardium or in the blood vessels perfusing the heart was unclear. The aim of the current study was to determine whether blood vessels per se can generate NO . from nitrite. In addition, since organic nitrates are relatively venoselective [2], and there is evidence that XO is also involved in their biotransformation to NO . [3], we wished to determine whether there are any differences between arteries and veins in the conversion of inorganic nitrite to NO.

Male Wistar rats (300-350 g, n = 6) were sacrificed and aortae and vena cavae were collected immediately. Tissues were homogenized, centrifuged and supernatants collected. The effect of these supernatant samples on NO . generation from nitrite (10-100  µm) in a 10 ml reaction chamber was assessed using ozone chemiluminescence. The mechanisms involved were investigated by incubation (30 min pretreatment) of tissue samples with various specific inhibitors.

Aorta and vena cava generated NO . from nitrite under ischaemic conditions, in a hypoxia, increasing acidity (from 7.4 to 5), and nitrite concentration-dependent manner. Addition of aorta or vena cava supernatant to nitrite (100  µm) at pH 5.5 increased NO . production from 214 ± 24 and 261 ± 76 to 826 ± 135 and 859 ± 182 pmol g^-1 tissue s^-1 respectively in hypoxia, an effect significantly attenuated in normoxic conditions: 423 ± 46 and 505 ± 118 pmol g^-1 s^-1 respectively (P < 0.01 both vessels, n = 6). Boiling of the supernatant, prior to incubation with nitrite, reduced the levels of measured NO by approximately 47% (P < 0.001, n = 6). Whilst the NO . synthase inhibitor N w-nitro- l-arginine methyl ester ( l-NAME) (300 µm, n = 5) had no effect, NO . production was attenuated by the XO inhibitors allopurinol (100 µm) and BOF-4272 (10 µ m) by about 20% in both cases (P < 0.05, n = 6), but increased by the XO substrate NADH (1 m m, n = 5) by 35% (P < 0.01). A fluorometric XO assay revealed similar XO activity between aorta and vena cava (39 ± 5.6 and 52 ± 9.6 pmol g^-1 s^-1 respectively, n = 6).

These studies demonstrate that the capacity to generate NO from nitrite is dependent on XO activity, hypoxia and pH rather than vessel type.

1. Webb A, et al. Proc Natl Acad Sci USA 2004; 101: 13683.
2. MacAllister RJ, et al. J Pharmacol Exp Ther 1995; 273: 154.
3. Millar TM, et al. FEBS Lett 1998; 427: 225.

AW was supported by the Research Advisory Board of Barts & The London .