Abstract
S-nitrosothiols have been implicated in the etiology of various pulmonary diseases. Many of these diseases display gender preferences in presentation or altered severity that occurs with puberty, the mechanism by which is unknown. Estrogen has been shown to influence the expression and activity of endothelial nitric oxide synthase (eNOS) which is associated with increased S-nitrosothiol production. The effects of gender hormones on the expression and activity of the de-nitrosylating enzyme S-nitrosoglutathione reductase (GSNO-R) are undefined. This report evaluates the effects of gender hormones on the activity and expression of GSNO-R and its relationship to N-acetyl cysteine (NAC)-induced pulmonary hypertension (PH). GSNO-R activity was elevated in lung homogenates from female compared to male mice. Increased activity was not due to changes in GSNO-R expression, but correlated with GSNO-R S-nitrosylation: females were greater than males. The ability of GSNO-R to be activated by S-nitrosylation was confirmed by: 1) the ability of S-nitrosoglutathione (GSNO) to increase the activity of GSNO-R in murine pulmonary endothelial cells and 2) reduced activity of GSNO-R in lung homogenates from eNOS−/− mice. Gender differences in GSNO-R activity appear to explain the difference in the ability of NAC to induce PH: female and castrated male animals are protected from NAC-induced PH. Castration results in elevated GSNO-R activity that is similar to that seen in female animals. The data suggest that GSNO-R activity is modulated by both estrogens and androgens in conjunction with hormonal regulation of eNOS to maintain S-nitrosothiol homeostasis. Moreover, disruption of this eNOS-GSNO-R axis contributes to the development of PH.
Highlights
S-nitrosylation, a redox-based modification of a cysteine thiol by nitric oxide, is a post translational modification that can alter a protein’s function
GSNO-R activity measured by liquid chromatography/mass spectroscopy (LC/MS) was significantly higher in adult (10–12 w) female animals compared to their corresponding adult male counterparts (Figure 1A)
Similar gender specific differences (2–3 fold) were detected when GSNO-R activity was measured by GSNO-dependent NADH consumption or modified Saville Assay (Tables 1,2,3) In contrast, no significant differences in GSNO-R activity were seen in the lungs of young (4 w) male and female animals (Figure 1B)
Summary
S-nitrosylation, a redox-based modification of a cysteine thiol by nitric oxide, is a post translational modification that can alter a protein’s function. Mechanisms that control the addition and/or removal of the NO group from cysteine thiols are essential in determining the net effect of this modification. Formation of endogenous S-nitrosothiols can be mediated through: 1) the activity of any one of the nitric oxide synthase (NOS) isoforms, 2) oxidative reactions generating nitrosative species (for example, Fe+3NO, N2O3,) or 3) transnitrosative reactions (NO+-transfer) [1,2,3]. The production of S-nitrosothiols is opposed by mechanisms mediating de-nitrosylation which can: 1) occur non-enzymatically via homolytic or heterolytic cleavage, 2) be catalyzed by transition metal ions and reactive oxygen species, or 3) occur through enzymatic degradation [1,4]. The primary substrate for GSNO-R is GSNO [5,6], the levels of other S-nitrosylated-proteins are affected indirectly through altered transnitrosation equilibria with GSNO
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