Glutathione (GSH) and the GSH synthesis gene Gclm modulate vascular reactivity in mice

Abstract

Oxidative stress has been implicated in the development of vascular disease and in the promotion of endothelial dysfunction via the reduction in bioavailable nitric oxide (NO()). Glutathione (GSH) is a tripeptide thiol antioxidant that is utilized by glutathione peroxidase (GPx) to scavenge reactive oxygen species such as hydrogen peroxide and phospholipid hydroperoxides. Relatively frequent single-nucleotide polymorphisms (SNPs) within the 5' promoters of the GSH synthesis genes GCLC and GCLM are associated with impaired vasomotor function, as measured by decreased acetylcholine-stimulated coronary artery dilation, and with increased risk of myocardial infarction. Although the influence of genetic knockdown of GPx on vascular function has been investigated in mice, no work to date has been published on the role of genetic knockdown of GSH synthesis genes on vascular reactivity. We therefore investigated the effects of targeted disruption of Gclm in mice and the subsequent depletion of GSH on vascular reactivity, NO() production, aortic nitrotyrosine protein modification, and whole-genome transcriptional responses as measured by DNA microarray. Gclm(-/+) and Gclm(-/-) mice had 72 and 12%, respectively, of wild-type (WT) aortic GSH content. Gclm(-/+) mice had a significant impairment in acetylcholine (ACh)-induced relaxation in aortic rings as well as increased aortic nitrotyrosine protein modification. Surprisingly, Gclm(-/-) aortas showed enhanced relaxation compared to Gclm(-/+) aortas, as well as increased NO() production. Although aortic rings from Gclm(-/-) mice had enhanced ACh relaxation, they had a significantly increased sensitivity to phenylephrine (PE)-induced contraction. Alternatively, the PE response of Gclm(-/+) aortas was nearly identical to that of their WT littermates. To examine the role of NO() or other potential endothelium-derived factors in differentially regulating vasomotor activity, we incubated aortic rings with the NO() synthase inhibitor L-NAME or physically removed the endothelium before PE treatment. L-NAME treatment and endothelium removal enhanced PE-induced contraction in WT and Gclm(-/+) mice, but this effect was severely diminished in Gclm(-/-) mice, indicating a potentially unique role for GSH in mediating vessel contraction. Whole-genome assessment of aortic mRNA in Gclm(-/-) and WT mice revealed altered expression of genes within the canonical Ca(2+) signaling pathway, which may have a role in mediating these observed functional effects. These findings provide additional evidence that the de novo synthesis of GSH can influence vascular reactivity and provide insights regarding possible mechanisms by which SNPs within GCLM and GCLC influence the risk of developing vascular diseases in humans.

FIGURE 1

Protein level of aortic GCLC and GCLM normalized to β actin as measured by western blot within WT, Gclm^−/+, and Gclm^−/− mice. Nine aortas were collected from each genotype, 3 aortas from each genotype were combined and homogenized together. Bars represent means from an n of 3, each n representing 3 aortas. All error bars in figures represent standard error of the mean (SEM). * & *** = Significant difference from the matched control at p-values of < 0.05 and 0.001, respectively.

FIGURE 2

a) Total aortic GSH (GSH+GSSG) measured by HPLC and normalized to protein level within WT, Gclm^−/+, and Gclm^−/− mice. b) Total aortic glutathione disulfide (GSSG) measured by HPLC and normalized to protein level within WT, Gclm^−/+, and Gclm^−/− mice. c) %GSSG of total aortic GSH (GSH+GSSG) measured by HPLC and normalized to protein level within WT, Gclm^−/+, and Gclm^−/− mice. d) ΔE_GSSG/2GSH calculated from aortic tissue, assuming pH of 7.4 and temperature of 37°C. Each GSH and GSSG measure was made in a homogenate of 3 combined aortas from the same genotype. An n of 3 WT, 3 Gclm^−/+, and 5 Gclm^−/− was reached. All error bars in figures represent standard error of the mean (SEM). * & *** = Significant difference from the matched control at p-values of < 0.05 and 0.001, respectively.

FIGURE 3

Acetylcholine- (a) and sodium nitroprusside- (b) stimulated aortic ring relaxation in 10 WT, 12 Gclm^−/+, and 9 Gclm^−/− mice. Vascular reactivity was analyzed by repeated-measurement 2-way ANOVA. Concentration-response curves were fitted with a nonlinear regression program (GraphPad Prism) to obtain values of maximal effect, which were compared by 1-way ANOVA.

FIGURE 4

Production of nitric oxide (NO•) as measured by NO-Fe(DETC)2 spin trap and ESR spectroscopy and further normalized to protein following 5 μM ACh-stimulation for 90 min in aortas from WT, Gclm^−/+, and Gclm^−/− mice in both raw data (a) and as fold change to WT by assay (b). Three aortas were combined together for each n, and an n of 5 was obtained for each genotype. Statistical significance was determined by T-Test between fold change by day between Gclm^−/+ and Gclm^−/− aortas.

FIGURE 5

Production of aortic cGMP as measured by enzyme immunoassay (EIA) normalized to tissue weight. Whole aortas were collected from male WT, Gclm^−/+, and Gclm^−/− mice and one cGMP measure was made per aorta, and an n of 8 was obtained per genotype.

FIGURE 6

a) Brightfield and fluorescent images of representative cross sections taken from the aortas of WT, Gclm^−/+, and Gclm^−/− mice. Red fluorescence, detected by immunofluorescence in the emission range of 600-700nm, represents positive nitrotyrosine staining. a) Quantification of mean fluorescence intensity, averaging 4 sections per aorta, in 3 aortas per genotype. Statistical significance was observed by T-Test comparing WT to Gclm^−/+ aortas.

FIGURE 7

Phenylephrine (PE)-stimulated contraction of aortic rings from 10 WT, 12 Gclm^−/+, and 9 Gclm^−/− mice as both total force (a) and as % of total K-PSS contraction (b). Vascular reactivity was analyzed by repeated-measurement 2-way ANOVA. Concentration-response curves were fitted with a nonlinear regression program (GraphPad Prism) to obtain values of maximal effect, which were compared by 1-way ANOVA.

FIGURE 8

Differences between untreated aortic rings and either L-Name treated or endothelium removed aortic rings in phenylephrine (PE)-stimulated contraction of aortas from 5 WT (a), 7 Gclm^−/+ (b), and 5 Gclm^−/− (c) mice, as measured by %KPSS total contraction. Statistical analysis between L-NAME and endothelium removed effects were determined by Two-way ANOVA (GraphPad Prism).

FIGURE 9

Differences between untreated aortic rings and either L-Name treated (panels a and b) or endothelium removed aortic rings (panels c and d) following phenylephrine (PE)-stimulated contraction. a) comparing aortas from 5 WT and 7 Gclm^−/+ following L-NAME treatment, b) 5 WT and 5 Gclm^−/− following L-NAME treatment, c) 5 WT and 7 Gclm^−/+ following endothelium removal, and d) 5 WT and 5 Gclm^−/− following endothelial removal as measured by %KPSS total contraction. Statistical analysis between L-NAME and endothelium removed effects were determined by Two-way ANOVA (GraphPad Prism).

FIGURE 10

Venn Diagram indicating the number of gene selected for by microarray using criteria of an unadjusted p-value of <0.05 and a |fold| difference >1.5. The intersection of these circles represents the number of genes that have both been selected for in Gclm^−/− vs WT comparison and Gclm^−/+ vs WT comparison.

FIGURE 11

Ingenuity Pathway Analysis results. The top ten canonical pathways dysregulated for the (a) Gclm^−/− vs WT and (b) Gclm^−/+ vs WT comparisons are shown. Pathways were ranked by p-value significance using genes selected by pre-established selection criteria.