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.

Non-heme (Fe3+) in the lung increases with age in both humans and rats.

Increasing non-heme iron concentrations in host tissues are potentially significant, because they can be associated with an increased risk of injury including infections, fibrosis, and neoplasms. We tested the hypothesis that non-heme (Fe3+) in the lung increases with age in both humans and rats. Human tissue was collected at autopsy before fixation occurred. The total number of specimens was 131 with 78 nonsmokers and 53 smokers. Tissue was hydrolyzed in 3 N hydrochloric acid and 10% trichloroacetic acid. Supernatant (Fe3+) was measured with a thiocyanate assay. Non-heme (Fe3+) increased with age in nonsmokers. The correlation coefficient between lung (Fe3+) and age in the nonsmokers was 0.58 (p < 0.0001). Iron stains were negative, whereas those for ferritin demonstrated increased uptake with aging. Smokers had significantly greater non-heme (Fe3+) relative to nonsmokers (101.1 and 46.0 micromol/L respectively; T = 11.44, p < 0.0001). Lung non-heme (Fe3+) in smokers also increased with age (r = 0.75; p < 0.0001). Iron stains demonstrated uptake in the proximity of retained pigmented material. Ferritin stains demonstrated intense uptake in both the macrophages and the airway and alveolar epithelium of smokers. An animal model was also analyzed for an effect of aging on lung non-heme (Fe3+). At specified times between 30 and 186 days of age, rats (n = 48) were anesthetized and exsanguinated, and the lungs were excised. In rats, similar to humans, a positive correlation was seen between lung non-heme (Fe3+) and age (r = 0.73; p = 0.007). Stains for iron in rat lung were uniformly negative, whereas those for ferritin demonstrated increased uptake by airway and alveolar epithelium in older rats. We conclude that non-heme (Fe3+) in lung tissue increases with age in both humans and rats. Elevations in lung non-heme (Fe3+) could contribute to an increased incidence of pneumonias, pulmonary fibrosis, and bronchogenic carcinoma observed among older individuals.

Lung inflammation after exposure to nonfibrous silicates increases with chelatable [Fe3+].

Lung exposures to complexes of coordinated iron can be associated with a neutrophilic alveolitis. We tested the hypothesis that lung inflammation after intratracheal instillation of mineral oxides in rats increases with surface-complexed [Fe3+]. The 10 mineral oxides employed had measurable [Fe3+] complexed to the dust surface. The metal was incompletely coordinated, as demonstrated by the ability of the particles to catalyze electron transfer and generate thiobarbituric acid (TBA) reactive products of deoxyribose. After exclusion of those silicates containing structural iron within the crystal lattice, there was a significant correlation between the concentration of chelatable metal and TBA-reactive products (r = 0.82; p = .04). Four days after intratracheal instillation of the 10 mineral oxide particles into rats, lavage neutrophils and protein were significantly increased for all dusts compared to injected saline. Among those dusts with no structural iron, the correlation between chelatable iron concentrations and percentage neutrophils did not reach significance (r = 0.73; p = .10), but that between metal and lavage protein did (r = 0.80; p = .05). We conclude that (1) mineral oxides complex iron cations at the surface, (2) in vitro measures of oxidant generation increase with the concentration of surface iron among those dusts with no structural iron, and (3) acute inflammation following introduction of these particles into the lower respiratory tract also increases with surface iron concentrations among those mineral oxides with no structural iron.