No abstract

1 The vasodilator properties and photochemical decomposition of two synthetic iron-sulphur-nitrosyl clusters (cluster A: [Fe4S4(NO)4], tetranitrosyl-tetra-j3-sulphido-tetrahedro-tetrairon; and B:[Fe4S3 (NO)7V-1, heptanitrosyl-tri-u3-thioxotetraferrate(-1)) have been investigated. Experiments were carried out on isolated, internally-perfused segments of rat tail artery.2 Bolus injections (10 Ml) of A or B (>0.25 mM) delivered into the internal perfusate generated sustained (or S-type) vasodilator responses, characterized by a persistent plateau of reduced tone due to NO released from clusters which enter and become trapped within endothelial cells. Clusters were therefore irradiated with visible laser light (Z=457.9 or 514.5 nm) either (a) in solution, while passing through a glass tube en route to the artery; or (b) when retained within the endothelium, by illuminating the artery directly during the plateau of an S-type response. Irradiation produced an additional vasodilator response, the magnitude of which depended upon wavelength and laser beam energy. 3 The nitric oxide synthase inhibitor, NG-monomethyl-L-arginine (100 jM), had no effect on lightinduced vasodilator responses. However, they were (a) blocked entirely by adding oxyhaemoglobin (5 ,uM) to the internal perfusate; and (b) greatly enhanced by the enzyme superoxide dismutase (150 u ml-').4 Photolysis of cluster B was measured by absorption spectroscopy and by detecting NO released with an electrochemical sensor. The photochemical reaction was found to be oxygen-dependent. The half-time for inactivation of cluster-derived NO was measured by interposing different lengths of tubing (i.e time delays) between the photolysis tube and NO sensor. The steady-state probe current decayed exponentially with increasing delay time, with a t112 of 21 s. The amplitudes of vasodilator responses of the tail artery also decreased exponentially by increasing the time delay (t112=58 s). Superoxide dismutase (150 u ml-') prevented this from happening, showing that 'inactivation' of cluster-derived NO was caused by reaction with superoxide anions formed during photolysis. 5 We conclude that potentiation of vasodilator responses to iron-sulphur-nitrosyl clusters by visible light is due to an oxygen-dependent photochemical reaction which accelerates the release of ligated nitrosyl groups as free NO. Based on our measurements, we estimate that ca 100 pM NO is sufficient to produce a just-detectable additional vasodilatation and that the ED50 dose is ca 3.7 nM.

1 A study has been made of the eect of neocuproine, a speci®c Cu(I) chelator, on vasodilator responses of rat isolated perfused tail artery to two nitrosothiols: S-nitroso-N-acetyl-D,L-penicillamine (SNAP) and S-nitroso-glutathione (GSNO). 2 Bolus injections (10 ml) of SNAP or GSNO (10 77 ± 10 73 M) were delivered into the lumen of perfused vessels pre-contracted with sucient phenylephrine (1 ± 7 mM) to develop pressures of 100 ± 120 mmHg. Two kinds of experiment were made: SNAP and GSNO were either (a) pre-mixed with neocuproine (10 74 M) and then injected into arteries; or (b) vessels were continuously perfused with neocuproine (10 75 M) and then injected with either pure SNAP or GSNO. 3 In each case, neocuproine signi®cantly attenuated vasodilator responses to both nitrosothiols, although the nature of the inhibitory eect diered in the two types of experiment. We conclude that the ability of exogenous nitrosothiols to relax vascular smooth muscle in our ex vivo model is dependent upon a Cu(I) catalyzed process. Evidence is presented which suggests that a similar Cu(I)-dependent mechanism is responsible for the release of NO from endogenous nitrosothiols and that this process may assist in maintaining vasodilator tone in vivo.

1 Photorelaxation of vascular smooth muscle (VSM) is caused by the release of nitric oxide (NO) from a ®nite 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 in¯uence the extent to which the store is regenerated in the dark. 2 Isolated rat tail arteries (RTAs) were perfused internally with Krebs solution containing phenylephrine (PE; mean [PE]+s.e.mean: 5.78+0.46 mM) and periodically exposed to laser light (l=514.5 nm, 6.3 mW cm 72 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 g-glutamylcysteine synthetase (three injections, 100 mg kg 71 at 8 h intervals); or (b) isolated RTAs that were perfused ex vivo with oxothiazolidine (OXO), a precursor of cysteine (10 74 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; P50.01). 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; P50.01). A hyperbolic curve ®t 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. 3 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 (P50.05) while treatment of isolated vessels with OXO resulted in a 31% increase above control levels (P50.05). 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 ultimately from endothelium-derived NO. The possible physiological signi®cance of a photolabile store of NO in VSM is discussed brie¯y.

Nicotinamide (NA) is currently entering clinical trials as a radiosensitizer. A major component of its activity is the improvement of tumour oxygenation resulting from a reduction in microregional ischaemia. NA is known to reduce arterial blood pressure in rodents, suggesting a vascular component in its mechanism of action. We have used an ex vivo system to study the direct action of NA on the contractile properties of vascular smooth muscle. Isolated pieces of rat tail artery were internally perfused with Krebs' solution at a constant flow rate so that constriction of the arterial smooth muscle could be measured as an increase in perfusion pressure. Transient vasoconstrictor responses lasting up to 10 min were induced with bolus injections (10 microliters) of phenylephrine, at concentrations ranging from 10(-5) to 10(-2) M, into the internal perfusate whereas a constant increase in vasoconstrictor tone, giving perfusion pressures of 43-84 mmHg, was induced by constantly perfusing with PE (5 x 10(-6) M) or raising the K+ concentration of the Krebs' solution to 122 mM. The addition of NA to the perfusate significantly reduced the size of the transient vasoconstrictor responses in a dose-dependent manner and induced the precontracted vessels to relax. This action of NA could not be blocked either by N omega-nitro-L-arginine methyl ester (L-NAME), indomethacin or propranolol. We conclude that direct effects on supplying blood vessels probably contribute to the oxygenating action of NA in tumours, though the precise mechanism remains obscure.