Inhaled nitric oxide alleviates hyperoxia suppressed phosphatidylcholine synthesis in endotoxin-induced injury in mature rat lungs

Abstract

Background: We investigated efficacy of inhaled nitric oxide (NO) in modulation of metabolism of phosphatidylcholine (PC) of pulmonary surfactant and in anti-inflammatory mechanism of mature lungs with inflammatory injury.

Methods: Healthy adult rats were divided into a group of lung inflammation induced by i.v. lipopolysaccharides (LPS) or a normal control (C) for 24 h, and then exposed to: room air (Air), 95% oxygen (O), NO (20 parts per million, NO), both O and NO (ONO) as subgroups, whereas [3H]-choline was injected i.v. for incorporation into PC of the lungs which were processed subsequently at 10 min, 4, 8, 12 and 24 h, respectively, for measurement of PC synthesis and proinflammatory cytokine production.

Results: LPS-NO subgroup had the lowest level of labeled PC in total phospholipids and disaturated PC in bronchoalveolar lavage fluid and lung tissue (decreased by 46-59%), along with the lowest activity of cytidine triphosphate: phosphocholine cytidylyltransferase (-14-18%) in the lungs, compared to all other subgroups at 4 h (p < 0.01), but not at 8 and 12 h. After 24-h, all LPS-subgroups had lower labeled PC than the corresponding C-subgroups (p < 0.05). LPS-ONO had higher labeled PC in total phospholipids and disaturated PC, activity of cytidylyltransferase, and lower activity of nuclear transcription factor-kappaB and expression of proinflammatory cytokine mRNA, than that in the LPS-O subgroup (p < 0.05).

Conclusion: In LPS-induced lung inflammation in association with hyperoxia, depressed PC synthesis and enhanced proinflammatory cytokine production may be alleviated by iNO. NO alone only transiently suppressed the PC synthesis as a result of lower activity of cytidylyltransferase.

Figure 1

Synthesis of total phospholipids (TPL) in the mature rat lungs. The amounts of radiolabeled TPL (cpm/g body weight) recovered from bronchoalveolar lavage fluid (BALF), and in the total lungs [i.e. in both lung tissue after lavage and BALF], were determined in lipopolysaccharides (LPS)-treated (black diagrams) and non-treated control (C, white) rats. Definition of subgroups (n = 8 each): Air, room air; ONO, 95% oxygen and nitric oxide in 20 parts per million; O, 95% oxygen; and NO, nitric oxide 20 parts per million. Definition of each subset figures: A-B, 4 h; C-D, 24 h; A and C, [³H] TPL in BALF; B and D, [³H] TPL in the total lungs. Values are means ± SD. §§ p < 0.01 versus all other subgroups; # p < 0.05, ## p < 0.01 versus the corresponding C subgroup.

Figure 2

Cytidine triphosphate: phosphocholine cytidylyltransferase (CCT) activity in lung homogenates, microsomes, and cytosol at 24 h of different gas exposure in control (white diagram) and lipopolysaccharides-treated (black) rats. Definition of the subgroups see Fig. 1 legends. Definition of the subset figures: A in the presence of lipid activator; B and C, in the absence of the lipid activator. Values are means ± SD in each subgroup (n = 8) and expressed as nmol/min/mg protein. * p < 0.05, ** p < 0.01 versus LPS-O subgroup; ## p < 0.01, versus the corresponding C subgroups.

Figure 3

Protein contents of cytidine triphosphate: phosphocholine cytidylyltransferase (CCT) in lung homogenates (A) and microsomes (B) at 24 h of different gas exposure in control (white diagram) and lipopolysaccharides-treated (black) rats. Definition of the subgroups see Fig. 1 legends. Western blotting for CCT protein quantitated with densitometric analysis of the autoradiograms. Values are means ± SD in each subgroup (n = 6) and expressed as arbitrary units. * p < 0.05, ** p < 0.01 versus LPS-O subgroup; ## p < 0.01, versus the corresponding C-subgroups.

Figure 4

Photomicrographs of histological findings in the animal lungs at 48 h of endotoxin exposure corresponding to 24 h of different gas exposure in control (C) and lipopolysaccharides (LPS)-treated rats. Definition of the subgroups see Fig. 1 legends. All the lungs were fixed by pulmonary arterial perfusion at deflation pressure of 10 cm H₂O for 30 min. Definition and characteristics of subset figures: A, C-Air, well aerated alveoli; B C-ONO, nearly normal alveolar structure alternative with over expanded alveoli; C, C-O, mild atelectatic alveoli; D, C-NO, nearly normal alveolar structure alternative with over expanded alveoli; E, LPS-Air, severe inflammation and edema; F LPS-ONO, severe inflammation and collapsed alternative with over expanded alveoli; G, LPS-O, very severe inflammation and collapse of alveoli; H, LPS-NO, mild collapse of alveoli. Hematoxylin and eosin staining; original magnification ×100.

Figure 5

Measurement of nuclear transcription factor κB (NF-κB) in the lung tissue at 4 (A) and 24 (B) h of different gas exposure in control (white diagram) and lipopolysaccharides-treated (black) rats. Definition of the subgroups see Fig. 1 legends. NF-κB DNA-binding activity was measured with electrophoretic mobility shift assay. The values from densitometry of bands are means ± SD (n = 6) and expressed as arbitrary units. ++ p < 0.01 versus LPS-Air; ** p < 0.01 versus LPS-O; ## p < 0.01 versus corresponding C subgroup.

Figure 6

Measurement of expression of proinflammatory cytokines of the lung tissue. Definition of the subgroups see Fig. 1 legends. Definition of subset figures at 4 and 24 h, respectively, of different gas exposure in control (white diagram) and lipopolysaccharides-treated (black) rats: A-B, tumor necrosis factor alpha (TNF-α) mRNA; C-D: cytokine-induced neutrophil chemoattractant-1 (CINC-1) mRNA; E-F: interleukin-10 (IL-10) mRNA. Values are means ± SD (n = 6) and expressed as arbitrary units. + p < 0.05, ++ p < 0.01 versus LPS-Air; ** p < 0.01 versus LPS-O; ## p < 0.01 versus corresponding C subgroup.

Figure 7

Measurement of activity of myeloperoxidase (MPO) and malondialdehyde (MDA). Definition of the subgroups see Fig. 1 legends. Definition of subset figures at 4 and 24 h, respectively, of different gas exposure in control (white diagram) and lipopolysaccharides-treated (black) rats: A-B, MPO; C-D, MDA. Values are means ± SD (n = 6). ++ p < 0.01 versus LPS-Air; * p < 0.05, ** p < 0.01 versus LPS-O; ## p < 0.01 versus corresponding C subgroup.