Selective modifiers of glutathione biosynthesis and 'repriming' of vascular smooth muscle photorelaxation.
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 temporarily but then recovers slowly as the photosensitive store is reconstituted in the dark. The recovery process, termed repriming, displays an absolute requirement for endothelium-derived NO and is inhibited by pre-treating arteries with ethacrynic acid, a thiol-alkylating agent. Here we demonstrate that agents that up- or down-regulate glutathione (GSH) biosynthesis influence the extent to which the store is regenerated in the dark. Isolated rat tail arteries (RTAs) were perfused internally with Krebs solution containing phenylephrine (PE; mean [PE] +/- s.e.mean: 5. 78+/-0.46 microM) and periodically exposed to laser light (lambda=514.5 nm, 6.3 mW cm(-2) for 6 min). Photorelaxations of control RTAs were compared with those from either (a) vessels taken from animals previously injected i.p. with buthionine sulphoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase (three 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 attenuated photorelaxations: the mean (+/-s.e.mean) amplitude of the response recorded 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 allowed 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 hyperbolic curve fit to repriming curves for BSO-treated and control arteries returned asymptote values (maximum photorelaxations) of (mean +/- s.e.mean) 24.2+/-3.2% and 55.2+/-8.5%, respectively. The level of GSH in RTA extracts was measured by high-pressure liquid chromatography (HPLC). Injecting animals with BSO decreased GSH to 85% of control levels (P<0.05) while treatment of isolated vessels with OXO resulted in a 31% increase above control levels (P<0.05). Thus, drug-induced changes in RTA GSH levels were positively correlated with altered photorelaxations. 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 ultimately from endothelium-derived NO. The possible physiological significance of a photolabile store of NO in VSM is discussed briefly.
Megson
I L
IL
Department of Biomedical Sciences, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD.
Holmes
S A
SA
Magid
K S
KS
Pritchard
R J
RJ
Flitney
F W
FW
D000818:Animals; D000963:Antimetabolites / Q000494:pharmacology; D000975:Antioxidants / Q000737:chemistry; D019328:Buthionine Sulfoximine / Q000494:pharmacology*; D005978:Glutathione / Q000031:analogs & derivatives* / Q000096:biosynthesis* / Q000378:metabolism / Q000502:physiology; D066298:In Vitro Techniques; D008297:Male; D009126:Muscle Relaxation / Q000187:drug effects*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000502:physiology; D009569:Nitric Oxide / Q000502:physiology; D009603:Nitroso Compounds / Q000378:metabolism; D010777:Photochemistry; D051381:Rats; D017208:Rats, Wistar; D026422:S-Nitrosoglutathione; D013623:Tail / Q000187:drug effects / Q000502:physiology; D013844:Thiazoles / Q000494:pharmacology*
D000963:Antimetabolites; D000975:Antioxidants; D009603:Nitroso Compounds; D013844:Thiazoles; D009569:Nitric Oxide; D019328:Buthionine Sulfoximine; D026422:S-Nitrosoglutathione; D005978:Glutathione