Release of the antioxidants ascorbate and urate from a nitrergically-innervated smooth muscle

1 The main object of the present study was to determine whether ascorbate, an antioxidant which has been shown to protect nitric oxide (NO) from attack by scavenger molecules, might be released from nitrergically-innervated smooth muscle; ascorbate release from the rat anococcygeus was measured by use of h.p.l.c. with electrochemical detection. 2 Incubation of rat anococcygeus muscles in normal physiological salt solution (PSS; 30 min) resulted in release of ascorbate into the bathing medium (7.7+/-0.9 nmol g(-1) tissue). This release was increased by 96% when muscles were incubated in high K+ (70 mM) PSS. The resting release of ascorbate was unaffected by tetrodotoxin (TTX; 1 mu M), omega-conotoxin GVIA (10 nM) or omission of calcium ions from the PSS (with addition of 0.2 mM EGTA), but all three procedures attenuated the increased release observed under depolarizing conditions. Resting release of ascorbate was unaffected by glutamate (100 mu M), aspartate (100 mu M), gamma-aminobutyric acid (100 mu M) or carbachol (50 mu M). 3 A second h.p.l.c. peak, which always preceded the ascorbate peak, was identified as urate. Urate release from the anococcygeus, following 30 min incubation in normal PSS, was 64.6+/-12.7 nmol g(-)1 tissue but, unlike ascorbate, urate release was unchanged in high K+ PSS. In functional experiments, urate (100-400 mu M) partially protected NO (15 mu M)-induced relaxations of the rat anococcygeus from inhibition by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO; 50 mu M), but not from inhibition by hydroquinone or duroquinone (both 100 mu M). 4 Muscles chemically sympathectomized with 6-hydroxydopamine (6-OHDA, 500 mu M; 2h) still exhibited release of ascorbate (2.5+/-0.4 nmol g(-1) tissue) and urate (22.2+/-2.9 nmol g(-1) tissue); in both cases the release was similar to that observed in time-matched control tissues not exposed to 6-OHDA. High K+ PSS produced a TTX-sensitive increase in release of ascorbate, but not urate, from 6-OHDA-treated muscles. 5 The results demonstrate that significant amounts of ascorbate and urate are released from the rat anococcygeus muscle. Ascorbate, but not urate, release appears to be enhanced by activation of nerves which are resistant to 6-OHDA pretreatment. Since both antioxidants can protect NO from attack by scavenger molecules, their release in nitrergically-innervated tissues may be important for the provision of the correct redox environment to allow NO to fulfill its proposed neurotransmitter role.