Gender Differences in S-Nitrosoglutathione Reductase Activity in the Lung

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

Female C57BL6/129SvEv mice do not develop PH with chronic, systemic administration of N-acetyl cysteine.

<p>C57Bl/129SvEv mice were untreated (N) or treated with 10 mg/ml NAC (NAC), 52 mM SNOAC (SNOAC) or hypoxia (H) for a period of 3 weeks. (A) Right ventricular pressure (RVP) and (B) right heart weight (expressed as right ventricular weight/left ventricular weight + septum weight (RV/LV+S) were determined. Female mice responded to only to SNOAC and hypoxia. * (<i>n = 19–25</i>, p<0.05).</p

Castration increases GSNO-R activity, protecting against NAC-induced increases in right heart weight and ventricular pressure.

<p>Male C57BL6/129SvEv mice with either intact gonads or castrated were treated with (white bars) or without NAC (black bars) for 3 weeks. (A) RVW and (B) RVP were measured. Castration eliminated the increase in both RVW and RVP (N = 11 intact, N = 10 castrated, p<0.001).</p

eNOS is required for GSNO-R activity.

<p>(A) GSNO-R activity was measured in the lung homogenates obtained from eNOS^−/− and wild type (C57Bl6) mice using a modified Saville Assay using 28 µM GSNO with no protein precipitation. GSNO-R activity was reduced by 50% in the eNOS^−/− lung homogenate compared to that seen in the wild type mice (n = 5–6, p<0.002). (B) Western blot analysis of GSNO-R protein present in wild type (C57Bl6) and eNOS^−/− mouse lungs. No significant differences were detected in GSNO-R protein levels (n = 3).</p

S-nitrosoglutatione activates GSNO-R.

<p>(A) Primary mouse lung endothelial cells were treated with 10 µM GSNO for 5 min and the activity of GSNO-R determined using a modified Saville Assay using 28 µM GSNO. GSNO-R activity was significantly increased after treatment of GSNO. (n = 3, p<0.011). (B) Cell lysates obtained from mouse lung endothelial cells were treated with or without 5 µM L-SNO-cysteine for 5 min. GSNO-R activity was measured by L-SNO-cysteine dependent NADH consumption. L-SNO-Cysteine resulted in a 2 fold increase in NADH consumption (n = 4, p<0.011).</p

GSNO-R activity and S-nitrosylation is elevated in the female mouse lung.

<p>(A) GSNO-R activity was measured in lung homogenates from male and female C57Bl6/129SvEv mice using LC/MS. GSNO-R activity is expressed as the amount of GSNO catabolized after 5 min. Activity present in female mouse lung homogenates was approximately two times that seen in the males (n = 20, p<0.003). (B) GSNO-R activity was measured by LC/MS in lung homogenates from young (4 w) C57Bl6/129SvEv sexually immature male and female mice. Gender differences in GSNO-R activity were not seen in the young animals (n = 5). (C) GSNO-R protein expression was determined in adult C57Bl6/129SvEv male and female lung homogenates. Protein expression was not significantly different between genders. (D) Lung homogenates from C57Bl6/129SvEv male and female mice were subjected to biotin switch to determine if GSNO-R was S-nitrosylated in vivo. Lung homogenates from both male and female animals demonstrate the presence of GSNO-R S-nitrosylation. However, the extent of S-nitrosylation was greater in the female animals. Incubation of the lung homogenates with mercuric chloride reduced the abundance of S-nitrosylated GSNO-R.</p

Comparison of Methods used to Measure NADH-Dependent GSNO-R Activity.

<p>Lungs were harvested from adult (10–12 w) C57Bl6/129SeEv mice. Lung homogenates were subjected to NADH-dependent GSNO-R activity. The assay was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014007#s4" target="_blank">Methods</a>. The effect of altering the concentration of GSNO in the assay and the precipitation of protein prior to measurement were compared in the same samples (n = 4). Note that higher activity was obtained in the absence of protein precipitation for both GSNO concentrations. Highest GSNO-R activity was obtained in the presence of 100 µM GSNO. GSNO-R activity was found to be 2–3 fold higher in the lung homogenates from female animals regardless of the method used to measure activity.</p

GSNO-R Activity determined by GSNO-dependent NADH Consumption is greater in lung homogenates obtained from female mice.

<p>GSNO-R activity was measured in lung, liver, and kidney homogenates obtained from adult (10–12 w) male and female mice using a GSNO-dependent NADH Consumption assay. NADH consumption was evaluated in homogenates (0.3 µg) in the absence or presence of 100 µM GSNO. Gender specific differences were seen in the lung homogenates (n = 4). No gender specific differences were seen with either the liver or the kidney homogenates (n = 2–4).</p

Estrogen Activation of GSNO-R is eNOS dependent.

<p>(A) Abundance of eNOS present in lung homogenates from male and female C57Bl6/129SvEv mice was determined by Western blot analysis using antibodies directed against eNOS and β-actin. eNOS protein levels were greater in lung homogenates of female animals. (B) Mouse lung endothelial cells isolated from female mouse lungs were treated with or without 10 µM estrogen in the presence or absence of 100 µM L-NAME for 4 h. Estrogen resulted in an increase in GSNO-R S-nitrosylation. The increased in GSNO-R S-nitrosylation was abrogated by pretreatment with L-NAME.</p

Serum SNOAC levels are lower in female mice.

<p>Serum SNOAC was measured by mass spectrometry (MS) in C57BL6/129SvEv male and female mice treated with NAC. Left panel  =  LC chromatogram: Right panel =  MS spectrum. Serum from NAC-treated male (red) and female (green) mice had a SNOAC peak (m/z 193) that co-migrated with the ¹⁵N-SNOAC standard (black). No signal was seen in non-treated mice (blue). The amount of SNOAC was less in the female mice.</p