Mechanisms of nitric oxide synthase uncoupling in endotoxin-induced acute lung injury: role of asymmetric dimethylarginine

Abstract

Acute lung injury (ALI) is associated with severe alterations in lung structure and function and is characterized by hypoxemia, pulmonary edema, low lung compliance and widespread capillary leakage. Asymmetric dimethylarginine (ADMA), a known cardiovascular risk factor, has been linked to endothelial dysfunction and the pathogenesis of a number of cardiovascular diseases. However, the role of ADMA in the pathogenesis of ALI is less clear. ADMA is metabolized via hydrolytic degradation to l-citrulline and dimethylamine by the enzyme, dimethylarginine dimethylaminohydrolase (DDAH). Recent studies suggest that lipopolysaccharide (LPS) markedly increases the level of ADMA and decreases DDAH activity in endothelial cells. Thus, the purpose of this study was to determine if alterations in the ADMA/DDAH pathway contribute to the development of ALI initiated by LPS-exposure in mice. Our data demonstrate that LPS exposure significantly increases ADMA levels and this correlates with a decrease in DDAH activity but not protein levels of either DDAH I or DDAH II isoforms. Further, we found that the increase in ADMA levels cause an early decrease in nitric oxide (NO(x)) and a significant increase in both NO synthase (NOS)-derived superoxide and total nitrated lung proteins. Finally, we found that decreasing peroxynitrite levels with either uric acid or Manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin (MnTymPyp) significantly attenuated the lung leak associated with LPS-exposure in mice suggesting a key role for protein nitration in the progression of ALI. In conclusion, this is the first study that suggests a role of the ADMA/DDAH pathway during the development of ALI in mice and that ADMA may be a novel therapeutic biomarker to ascertain the risk for development of ALI.

Fig. 1

Superoxide levels in the mouse lung after LPS exposure. Relative superoxide levels were determined by electron paramagnetic resonance (EPR) in LPS-treated mouse lungs. There is a significant increase in superoxide radical generation 2- and 4 h after LPS exposure that is blocked by the addition of the NOS inhibitor, NG-monomethyl L-arginine (L-NMMA; 100 μM) or polyethylene glycol-superoxide dismutase (PEGSOD; 100 U/ml). There is no change in superoxide generation 12 h after LPS exposure. Values are means ± SE, N=6. *P<0.05 vs. no LPS, †P<0.05 vs. LPS alone.

Fig. 2

Lung tissue NO_x and tetrahydrobiopterin levels after LPS exposure. Tissue NO_x levels are significantly decreased 2 h after LPS exposure. However, NO_x levels were significantly increased 4- and 12-h after LPS exposure (A). Lung BH₄ levels were measured by HPLC. There was no change in BH₄ levels 2 h after LPS exposure, but BH₄ levels were significantly increased 4- and 12-h post LPS treatment (B). Values are mean±SE, N=5 for each group. *P<0.05 compared to no LPS, †P<0.05 compared to previous time point.

Fig. 3

NOS isoform protein levels in the mouse lung after LPS exposure. Protein levels for eNOS, nNOS, and iNOS were determined in lung tissues 2- and 4-h after LPS exposure by Western blot analysis using specific antisera raised against eNOS, nNOS, or iNOS respectively and re-probed with β-actin to normalize for loading. Representative Western blots are shown for eNOS (panel A), nNOS (panel B), and iNOS (panel C). There are no changes in eNOS or nNOS protein levels but iNOS protein levels are significantly increased 4 h after LPS exposure. Values are mean ± SE, N=5 for each group *P<0.05 compared to no LPS.

Fig. 4

LPS exposure leads to elevated ADMA and decreased DDAH activity in the mouse lung. ADMA levels and DDAH activity were analyzed by high-performance liquid chromatography (HPLC) in the mice lungs after LPS exposure. There was a progressive increase in ADMA levels after 2-, 4- and 12-h LPS exposure (A). Although there were no changes in either DDAH I (B) or DDAH II (C) protein levels there was a significant decrease in DDAH activity 2- and 4-h after LPS exposure (panel D). Values are mean ± SE, N=5 for each group, *P<0.05 compared to no LPS.

Fig. 5

Nitrated protein levels are increased in the mouse lung after LPS exposure. Protein levels for total nitrated proteins were determined in the mouse lung 4 h after LPS exposure. Homogenates (25 μg) were separated on a 4–20% denaturing polyacrylamide gel, electrophoretically transferred to PVDF-nitrocellulose membranes, and analyzed using specific antiserum raised against 3-NT residues. A representative image for the Western blot analysis for 3-NT protein levels is shown (A). The boxed area shows the region of total nitrated proteins that was used to quantify 3-NT levels. Densitometric analysis indicates that LPS exposure increases total nitrated proteins in the mouse lung (B). Values are mean ± SE, N=5. *P<0.05 vs. no LPS.

Fig. 6

Peroxynitrite scavenging decreases the LPS-mediated hyperpermeability in the mouse lung. Protein levels for total nitrated proteins were determined in the mouse lung 4 h after LPS exposure in the presence and absence of the ONOO– scavengers, uric acid and MnTymPyp. The presence of either uric acid and MnTymPyp significantly decreased total nitrated protein levels compared to that obtained with LPS alone (A) and this correlated with a significant decrease in Evans blue dye levels in the lungs indicating a decrease in lung permeability (B). Values are mean ± SE, N=5. *P<0.05 vs. no LPS, †P<0.05 vs. LPS alone.

Fig. 7

ADMA potentiates the decrease in transendothelial electrical resistance (TER) associated with VEGF exposure in human lung microvascualr endothelial cells. Confluent were exposed or not to ADMA (5 μM, 1 h). Cells were then exposed or not to VEGF (100 ng) and changes in TER across the endothelial cell monolayer were measured by ECIS. Although ADMA alone does not induce a change in TER there is a potentiation in the decrease in TER associated with VEGF exposure. Values are mean ± SE, N=5. *P<0.05 vs. untreated, †P<0.05 vs. VEGF alone.