Exposure to a hypoxic challenge increases ventilation in wild-type (WT) mice that diminish during the challenge (roll-off) whereas return to room air causes an increase in ventilation (short-term facilitation, STF). Since plasma and tissue levels of ventilatory excitant S-nitrosothiols such as S-nitrosoglutathione (GSNO) increase during hypoxia, this study examined whether (1) the initial increase in ventilation is due to generation of GSNO, (2) roll-off is due to increased activity of the GSNO degrading enzyme, GSNO reductase (GSNOR), and (3) STF is limited by GSNOR activity. Initial ventilatory responses to hypoxic challenge (10% O2, 90% N2) were similar in WT, GSNO+/− and GSNO−/− mice. These responses diminished markedly during hypoxic challenge in WT mice whereas there was minimal roll-off in GSNOR+/− and GSNOR−/− mice. Finally, STF was greater in GSNOR+/− and GSNOR−/− mice than WT mice (especially females). This study suggests that GSNOR degradation of GSNO is a vital step in the expression of ventilatory roll-off and that GSNOR suppresses STF.

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.

The aim of this study was to compare the ventilatory responses of C57BL6 female and male mice during a 15 min exposure to a hypoxic-hypercapnic (H-H) or a hypoxic (10% O2, 90% N2) challenge and subsequent return to room air. The ventilatory responses to H-H were similar in males and females whereas there were pronounced gender differences in the ventilatory responses during and following hypoxic challenge. In males, the hypoxic response included initial increases in minute volume via increases in tidal volume and frequency of breathing. These responses declined substantially (roll-off) during hypoxic exposure. Upon return to room-air, relatively sustained increases in these ventilatory parameters (short-term potentiation) were observed. In females, the initial responses to hypoxia were similar to those in males whereas roll-off was greater and post-hypoxia facilitation was smaller than in males. The marked differences in ventilatory roll-off and post-hypoxia facilitation between female and male C57BL6 mice provide evidence that gender is of vital importance to ventilatory control.

Exposure to hypoxia elicits an increase in minute ventilation that diminishes during continued exposure (roll-off). Brainstem Nmethyl-D-aspartate receptors (NMDARs) and neuronal nitric oxide synthase (nNOS) contribute to the initial hypoxia-induced increases in minute ventilation. Roll-off is regulated by platelet-derived growth factor receptor-b (PDGFR-b) and S-nitrosoglutathione (GSNO) reductase (GSNOR). S-nitrosylation inhibits activities of NMDAR and nNOS, but enhances GSNOR activity. The importance of S-nitrosylation in the hypoxic ventilatory response is unknown. This study confirms that ventilatory roll-off is virtually absent in female GSNOR 1/2 and GSNO 2/2 mice, and evaluated the location of GSNOR in female mouse brainstem, and temporal changes in GSNOR activity, protein expression, and S-nitrosylation status of GSNOR, NMDAR (1, 2A, 2B), nNOS, and PDGFR-b during hypoxic challenge. GSNOR-positive neurons were present throughout the brainstem, including the nucleus tractus solitarius. Protein abundances for GSNOR, nNOS, all NMDAR subunits and PDGFR-b were not altered by hypoxia. GSNOR activity and S-nitrosylation status temporally increased with hypoxia. In addition, nNOS Snitrosylation increased with 3 and 15 minutes of hypoxia. Changes in NMDAR S-nitrosylation were detected in NMDAR 2B at 15 minutes of hypoxia. No hypoxia-induced changes in PDGFR-b Snitrosylation were detected. However, PDGFR-b phosphorylation increased in the brainstems of wild-type mice during hypoxic exposure (consistent with roll-off), whereas it did not rise in GSNOR 1/2 mice (consistent with lack of roll-off). These data suggest that: (1) S-nitrosylation events regulate hypoxic ventilatory response; (2) increases in S-nitrosylation of NMDAR 2B, nNOS, and GSNOR may contribute to ventilatory roll-off; and (3) GSNOR regulates PDGFR-b phosphorylation.Keywords: hypoxic ventilatory response; neuronal nitric oxide synthase; N-methyl-D-aspartate receptor; platelet-derived growth factor receptor-b; S-nitrosoglutathione reductase Clinical RelevanceThis article reports that the change in S-nitrosylation status of key brainstem proteins may underlie the roll-off phase of the hypoxic ventilatory response. This has potential clinical applications for understanding how mechanisms that affect S-nitrosothiol bioavailability (i.e., S-nitrosoglutathione reductase) play a role in central breathing disorders.

S-nitrosoglutathione (GSNO) stabilizes the a-subunit of hypoxia inducible factor-1 (HIF-1) in normoxic cells, but not in the presence of PI3K inhibitors. In this report, the biochemical pathway by which GSNO alters PI3K/Akt activity to modify HIF-1 expression was characterized in Cos cells and primary pulmonary vascular endothelial cells. GSNO increased Akt kinase activity-and downstream HIF1a protein accumulation and DNA-binding activity-in a dose-and time-dependent manner. The PI3K inhibitors, wortmannin and LY294002, blocked these responses. Neither glutathione nor 8-bromo-cyclic GMP mimicked the GSNO-induced increases in Akt kinase activity. GSNO-induced Akt kinase activity and downstream HIF-1a stabilization were blocked by acivicin, an inhibitor of g2glutamyl transpeptidase (gGT), a transmembrane protein that can translate extracellular GSNO to intracellular S-nitrosocysteinylglycine. Dithiothreitol blocked GSNO-induced Akt kinase activity and HIF-1a stabilization. Moreover, the 39-phosphatase of phosphoinositides, PTEN (phosphatase and tensin homolog deleted on chromosome ten) was S-nitrosylated by GSNO in pulmonary arterial endothelial cells, which was reversed by dithiothreitol and ultraviolet light. Interestingly, the abundance of S-nitrosylated PTEN also correlated inversely with PTEN activity. Taken together, these results suggest that GSNO induction of Akt appears to be mediated by S-nitrosylation chemistry rather than classic NO signaling through guanylate cyclase/cGMP. We speculate that gGT-dependent activation of Akt and subsequent activation of HIF-1 in vascular beds may be relevant to the regulation of HIF-1-dependent gene expression in conditions associated with oxyhemoglobin deoxygenation, as opposed to profoundly low PO 2 , in the pulmonary vasculature.Keywords: phosphatidylinositol 3-kinase; hypoxia-inducible factor-1; S-nitrosothiol; g-glutamyl transpeptidase; PTEN Hypoxia-inducible factor (HIF)-1 promotes the expression of several genes that confer hypoxic tolerance through angiogenesis, erythropoeisis, vasodilation, and altered glucose metabolism. In hypoxia, the regulatory subunit of the HIF-1 heterodimer, HIF1a, is stabilized through decreased activity of prolyl hydroxylases that target the protein for degradation (1). In normoxia, HIF-1 can also be activated by mitogen-activated protein kinase (MAPK)-dependent processes initiated by growth factors (2-4) or S-nitrosoglutathione (GSNO) (5). Indeed, exposure to GSNO in normoxia results in the accumulation of HIF-1a protein. Of note, inhibitors of the phosphatidylinositol 39-kinase (PI3K)-initiated Akt activation prevent this effect, implicating this signaling pathway in the GSNO effect (6).Redox-associated modification of cysteine thiols by nitric oxide (NO) in biology can regulate the function of proteins (7-9). S-nitrosylated proteins are involved in numerous signaling pathways. Transnitrosation, the transfer of NO in the form of nitrosonium (NO 1 ) from one cysteine thiol to a second cysteine thiol, is believed to be responsible for m...