Selective modifiers of glutathione biosynthesis and 'repriming' of vascular smooth muscle photorelaxation

1 Photorelaxation of vascular smooth muscle (VSM) is caused by the release of nitric oxide (NO) from a finite molecular store that can be depleted by irradiating pre-contracted arteries with visible light. The ability of an ' exhausted' vessel to respond to a further period of illumination is lost te mporarily but then recovers slowly as the photosensitive store is reconstit uted in the dark. The recovery process, termed repriming, displays an absol ute requirement for endothelium-derived NO and is inhibited by pre-treating arteries with ethacrynic acid, a thiol-alkylating agent. Here we demonstra te that agents that up- or down-regulate glutathione (GSH) biosynthesis inf luence the extent to which the store is regenerated in the dark. 2 Isolated rat tail arteries (RTAs) were perfused internally with Krebs sol ution containing phenylephrine (PE; mean [PE]+/-s.e.mean: 5.78+/-0.46 mu M) and periodically exposed to laser light (lambda=514.5 nm, 6.3 mW cm(-2) fo r 6 min). Photorelaxations of control RTAs were compared with those from ei ther (a) vessels taken from animals previously injected i.p. with buthionin e sulphoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase (th ree injections, 100 mg kg(-1) at 8 h intervals); or (b) isolated RTAs that were perfused ex vivo with oxothiazolidine (OXO), a precursor of cysteine ( 10(-4) M OXO for 60 min). RTAs from BSO-treated animals exhibited attenuate d photorelaxations: the mean (+/-s.e.mean) amplitude of the response record ed after 72 min recovery in the dark was 12.4+/-1.6% versus 21.4+/-2.9% for control arteries (n=5; P<0.01). Conversely RTAs treated with OXO and allow ed to recover for a similar period showed enhanced photorelaxations, 32.6+/ -6.3% as compared to 21.4+/-2.9% for control arteries (n=5; P<0.01). A hype rbolic curve fit to repriming curves for BSO-treated and control arteries r eturned asymptote values (maximum photorelaxations) of(mean+/-s.e.mean) 24. 2+/-3.2% and 55.2+/-8.5%, respectively. 3 The level of GSH in RTA extracts was measured by high-pressure liquid chr omatography (HPLC). Injecting animals with BSO decreased GSH to 85% of cont rol levels (P<0.05) while treatment of isolated vessels with OXO resulted i n a 31% increase above control levels (P<0.05). Thus, drug-induced changes in RTA GSH levels were positively correlated with altered photorelaxations. 4 The results lead us to postulate that the photosensitive store in VSM is generated, at least in part, from intracellular GSH which becomes converted to S-nitrosoglutathione (GSNO) by nitrosating species that are formed ulti mately from endothelium-derived NO. The possible physiological significance of a photolabile store of NO in VSM is discussed briefly.