C57BL/6/129SvEv, C57BL/6, and eNOS^–/– male mice were maintained in normoxia (N, red; 21% O₂) or hypoxia (H, black; 10% O₂); or were treated with NAC (blue) or SNOAC (green) in their drinking water for 3 weeks. (A) Relative RV weight was determined as the ratio of the weight of the RV to the LV+S weight. (B) RV systolic pressures were measured in the closed chest using a Millar 1.4 F catheter/transducer. (C) Representative RV pressure (RVP) tracings (each = 1 s). (D) Lung section images from C57BL/6/129SvEv mice immunostained for von Willebrand factor and α-SMA to illustrate changes in muscularization after 3 weeks of exposure to normoxia, hypoxia, NAC, or SNOAC. Scale bar: 100 μm (applies to all panels). (E) Changes in muscularization in C57BL/6/129SvEv mice in the small (<80-μm) vessels from histological sections (as in D) counted by an observer blinded to the protocol. FM, fully muscular; PM, partly muscular, NM, nonmuscular. Significant increases in muscularization in each treatment group were seen in comparison to normoxic controls. Data are mean ± SEM. ^ζP < 0.02, *P < 0.001, ^†P < 0.003, by 1-way ANOVA followed by pairwise comparison, all compared with normoxic control.

The expression of fibronectin (A), HIMF (B), eNOS (C and D), VEGF-A (E), and endothelin (F) in whole-lung homogenates from NAC-treated mice was examined by immunoblot. Fold increase in density relative to MAPK (equal loading control) was determined for each condition. The increases in fibronectin, HIMF, and eNOS (n = 3–5 each) were significant. (G) Three weeks of NAC treatment also increased whole-lung mRNA, assayed relative to 18S RNA by RT-PCR, for HIMF and VEGF-A but not fibronectin or Glut-1 (n = 3 each). Time course analysis of NAC-treated mice (D) revealed that the increase in whole-lung eNOS mRNA (filled squares, left axis) preceded the increase in eNOS protein expression (open circles, right axis) but decreased by 3 weeks. *P < 0.05; ^#P < 0.01.

(A) The SNO_rbc in heparinized LV blood (black bars), measured by reductive chemiluminescence (11), was lower than normal following 3 weeks of treatment with 10 mg/ml NAC (n = 3–4 each). In the same mice, plasma SNOAC levels (gray bars; measured by MS) increased from undetectable to approximately 2 μM over the same time (*P < 0.05). (B) Serum SNOAC, measured by MS, formed in NAC-treated mice (3 weeks). Left: liquid chromatogram; right: MS spectrum. NAC-treated mice had a SNOAC peak (m/z 193; red) coeluting with ¹⁵N-labeled SNOAC standard (m/z 194; black) that was absent in untreated animals (green) and was not detected in NAC-treated mice after serum pretreatment with HgCl₂ to displace NO⁺ from the thiolate (blue). (C) Oxygenated erythrocytes were deoxygenated ex vivo (argon; ref. 11) in the presence of 100 μM NAC; supernatant SNOAC was measured by MS (above). SNOAC concentration increased with oxyhemoglobin (Oxy Hb) desaturation (co-oximetry: inset), being maximal at 59.3% saturation (blue), less at 77.2% saturation (green), and undetectable at 98% saturation. (D) SNOAC (filled circles) accumulated as the concentration of S-nitrosothiol–modified Hb (SNOHb; open circles) and oxyhemoglobin saturation (Hb SO₂; blue line) both decreased in heparinized whole blood using argon with 5% CO₂ (pH 7.3) in a tonometer. Both the increase in SNOAC and the loss of SNO_rbc between 0 and 20 minutes were significant (P < 0.01 by ANOVA followed by pairwise comparison to the maximum value; n = 3). ^#SNOAC levels were below the limit of detection when the oxyhemoglobin saturation was greater than 80%.

(A) One micromolar SNOAC, but not 50 μM NAC, treatment (4 hours each) increased intracellular S-nitrosothiol levels (assayed by Cu/cysteine chemiluminescence; ref. 11) in primary murine pulmonary endothelial cells (*P < 0.05 compared with SNOAC treatment). (B) Immunoblot showing increased Sp3 expression relative to MAPK in the whole-lung homogenates of mice treated for 3 weeks with 10 mg/ml NAC but not in those of control mice. By densitometry, this increase was significant (P < 0.01). (C) Paradoxically, however, NAC (50 μM; 4 hours) did not increase Sp3 expression relative to β-actin in primary murine pulmonary endothelial cells in vitro, while both SNOAC (1 μM; 4 hours) and hypoxia (10%; 4 hours) did.

(A) NAC treatment (10 mg/ml; 3 weeks) increased whole-lung HIF 1–DNA binding activity. Complexes were supershifted (ss) with anti–HIF 1β and eliminated with excess cold probe (P). (B) SNOAC (1 μM), like GSNO (5, 13, 38), increased normoxic HIF 1α expression in nuclear extracts isolated from primary murine pulmonary endothelial cells. NAC alone (50 μM) did not affect HIF 1α expression. β-Actin was used as a protein load control. (C) In BPAECs transfected with HA-tagged HIF 1α and dominant-negative His-6-Myc–tagged ubiquitin (DN-Ub), ubiquitinated proteins were isolated using a nickel column and immunoblotted for HIF 1α. Both hypoxia and GSNO (G; 10 μM) inhibited HIF 1α ubiquitination relative to normoxia. (D) In COS cells cotransfected with HA-tagged HIF 1α and FLAG-tagged pVHL, GSNO (10 μM) prevented the coimmunoprecipitation of HIF 1α with pVHL. (E) S-nitrosylation of pVHL by SNOAC (5 μM) in equal protein aliquots isolated from HeLa cells was identified by biotin substitution (49); in the absence of ascorbate, S-nitrosylated pVHL was not detected. (F) Similarly, SNOAC and GSNO (5 μM) increased pVHL S-nitrosylation in pVHL-overexpressing 786-O cells. (G) C162, but not C77, was identified by biotin substitution to be S-nitrosylated in BPAECs transfected with wild-type cysteine 77 to serine mutant (C77S), C162S, or combined C77S/C162S pVHL exposed to SNOAC (1 μM). Native pVHL and MAPK immunoblots represented the pVHL expression and protein load controls, respectively. All in vitro treatments were for 4 hours.