TABLE 3.
Impact of various agents on N2-mediated enhancement of MP production, iNOS activation, DCF-DA fluorescence, and FBE formation
Isolated murine neutrophils were exposed to air or air plus 690 kPa of N2. MPs/PMN reflects MP counts in suspensions first exposed to air versus 690 kPa of N2 for 30 min and then left in air at ambient pressure for 4-h incubations. NOS activity reflects pmol of [H3]citrulline produced when [H3]arginine was added immediately after 30-min air/N2 exposures, and cells were incubated for 1 h at ambient pressure in air. DCF fluorescence was assessed when DCF-DA was added to cell suspensions that were then exposed to air or 690 kPa of N2 for 10 min. Inhibitor values in the N2 column are all significantly different from N2 + PBS (p < 0.05). FBEs reflect pyrene actin fluorescence in arbitrary units/min in cells studied immediately after 30-min air or 690-kPa N2 exposures. All values are mean ± S.E. (n = number of independent trials (shown in parentheses)). KO, neutrophils from NOS-2 knock-out mice; 1400W, incubation with 1 mm 1400W during air/N2 exposure; Ebselen, incubation with 1 mm ebselen during the air/N2 exposure; UV, cells exposed to UV light for 5 min after the 30-min air/N2 exposure before readings taken; Cyto D, incubation with 5 μm cytochalasin D during the air/N2 exposure; Nox2ds, incubation with 10 μm Nox2ds during the air/N2 exposure; Scrmb-Nox2ds, incubations performed with 10 μm control, scrambled sequence peptide to Nox2ds; Rac-i, incubation with 50 μm NSC 23766 during the air/N2 exposure; Cont-si, cells incubated with control, scrambled sequence small inhibitory RNA for 24 h prior to the experiment; VASP-si, cells incubated with small inhibitory RNA to vasodilator-stimulated phosphoprotein for 24 h prior to the experiment; FAK-i, cells incubated with 20 μm PT 573228 during the air/N2 exposure; FAK-si, cells incubated with small inhibitory RNA to focal adhesion kinase for 24 h prior to the experiment; Flip-i, cells incubated with 200 μm SCH 28080 during the air/N2 exposure; Flip-si, cells incubated with small inhibitory RNA to flippase for 24 h prior to the experiment; Flop-i, cells incubated with 200 μm MK 571 during the air/N2 exposure; Flop-si, cells incubated with small inhibitory RNA to floppase for 24 h prior to the experiment; PDI-i, cells incubated with 30 μm quercetin-3-rutinoside during the air/N2 exposure, PDI-si, cells incubated with small inhibitory RNA to protein-disulfide isomerase for 24 h prior to the experiment; ND, no experiments performed with KO mice; fluor, fluorescence.
| Agent | MPs/PMN/h × 102 |
iNOS (pmol [H3]citrulline/h) |
DCF (fluor at 10 min × 10−1) |
FBEs (fluor/min) × 103 |
||||
|---|---|---|---|---|---|---|---|---|
| Air | N2 | Air | N2 | Air | N2 | Air | N2 | |
| PBS | 0.03 ± 0.02 (24) | 384 ± 21 (8)a | 0.19 ± 0.01 (21) | 1.76 ± 0.08 (24)a | 0.72 ± 0.09 (11) | 1213 ± 56.3 (12)a | 0.46 ± 0.05 (12) | 8.55 ± 0.40 (12)a |
| KO | 0.00 ± 0.00 (5) | 0.00 ± 0.00 (5) | 0 ± 0 (5) | 0.01 ± 0.03 (5) | ND | ND | 0.39 ± 0.06 (3) | 0.59 ± 0.16 (3) |
| 1400W | 0.00 ± 0.00 (4) | 0.13 ± 0.06 (5) | 0 ± 0 (8) | 0 ± 0 (8) | 0.93 ± 0.07 (3) | 74 ± 13.0 (3)a | 0.54 ± 0.12 (5) | 0.42 ± 0.08 (3) |
| Ebselen | 0.06 ± 0.02 (3) | 0.06 ± 0.04 (3) | 0.20 ± 0.001 (3) | 0.14 ± 0.04 (3) | 0.0 ± 0.0 (4) | 63.0 ± 48.1 (4) | 0.30 ± 0.01 (5) | 0.54 ± 0.12 (5) |
| UV | 0.02 ± 0.003 (4) | 0.05 ± 0.03 (4) | 0.20 ± 0.003 (5) | 0.10 ± 0.001 (4) | 0.95 ± 0.18 (3) | 100.4 ± 56.5 (3)a | 0.42 ± 0.02 (3) | 0.46 ± 0.11 (3) |
| Cyto D | 0.09 ± 0.003 (3) | 0.13 ± 0.06 (3) | 0.20 ± 0.008 (3) | 0.14 ± 0.05 (6) | 0.92 ± 0.19 (3) | 141.2 ± 30.3 (3)a | 0 ± 0 (3) | 0 ± 0 (3) |
| Nox2ds | 0.04 ± 0.02 (3) | 0.05 ± 0.03 (3) | 0.20 ± 0.001 (3) | 0.04 ± 0.01 (4) | 0.92 ± 0.19 (3) | 117.0 ± 48.4 (3)a | 0.40 ± 0.10 (4) | 0.53 ± 0.16 (4) |
| Scrmb-Nox2ds | 0.02 ± 0.003 (3) | 496 ± 12 (3)a | 0.18 ± 0.01 (3) | 1.57 ± 0.08 (5)a | 0.91 ± 0.22 (3) | 1139 ± 82.8 (4)a | 0.30 ± 0.02 (4) | 8.11 ± 0.25 (4)a |
| Rac-i | 0.08 ± 0.08 (3) | 0.16 ± 0.12 (3) | 0.21 ± 0.003 (4) | 0.31 ± 0.003 (4) | 0.0 ± 0.0 (4) | 76.0 ± 76.0 (4) | 0.34 ± 0.05 (5) | 0.41 ± 0.08 (4) |
| Cont-si | 0.03 ± 0.02 (4) | 405 ± 42 (4)a | 0.20 ± 0.001 (4) | 1.71 ± 0.04 (4)a | 0.81 ± 0.19 (4) | 1214 ± 51.1 (4)a | 0.44 ± 0.03 (4) | 8.61 ± 0.55 (4)a |
| VASP-si | 0.09 ± 0.17 (4) | 0.15 ± 0.07 (4) | 0.20 ± 0.001 (4) | 0.03 ± 0.001 (4) | 0.97 ± 0.22 (3) | 281.1 ± 81.4 (3)a | 0.40 ± 0.06 (3) | 0.57 ± 0.22 (3) |
| FAK-i | 0.04 ± 0.02 (3) | 0.07 ± 0.03 (3) | 0.20 ± 0.001 (3) | 0.18 ± 0.14 (3) | 1.00 ± 0.25 (3) | 236.8 ± 98.3 (3)a | 0.42 ± 0.04 (5) | 0.33 ± 0.03 (3) |
| FAK-si | 0.05 ± 0.02 (4) | 0.08 ± 0.04 (3) | 0.19 ± 0.002 (4) | 0.09 ± 0.002 (4) | 0.72 ± 0.12 (3) | 205.1 ± 58.3 (3)a | 0.40 ± 0.17 (3) | 0.67 ± 0.09 (3) |
| Flip-i | 0.07 ± 0.02 (3) | 0.16 ± 0.12 (3) | 0.20 ± 0.001 (4) | 0.19 ± 0.14 (4) | 4.2 ± 4.2 (4) | 45.8 ± 45.8 (4) | 0.42 ± 0.03 (3) | 0.35 ± 0.08 (3) |
| Flip-si | 0.02 ± 0.02 (4) | 0.06 ± 0.04 (4) | 0.20 ± 0.002 (3) | 0.16 ± 0.11 (4) | 0.98 ± 0.13 (3) | 222.4 ± 95.9 (3)a | 0.43 ± 0.03 (3) | 0.43 ± 0.03 (3) |
| Flop-i | 0.003 ± 0.01 (3) | 0.05 ± 0.02 (3) | 0.20 ± 0.001 (4) | 0.16 ± 0.11 (4) | 0.0 ± 0.0 (4) | 12.7 ± 33.9 (4) | 0.48 ± 0.09 (3) | 0.38 ± 0.06 (5) |
| Flop-si | 0.04 ± 0.04 (4) | 0.05 ± 0.04 (4) | 0.20 ± 0.001 (3) | 0.32 ± 0.16 (4) | 0.95 ± 0.10 (3) | 235.8 ± 97.3 (3)a | 0.47 ± 0.03 (3) | 0.53 ± 0.09 (3) |
| PDI-i | 0.02 ± 0.02 (3) | 0.01 ± 0.003 (3) | 0.19 ± 0.003 (4) | 0.24 ± 0.003 (4) | 0.57 ± 0.22 (3) | 146.0 ± 55.8 (3)a | 0.39 ± 0.04 (3) | 0.53 ± 0.12 (4) |
| PDI-si | 0.04 ± 0.03 (4) | 0.06 ± 0.06 (4) | 0.20 ± 0.003 (3) | 0.12 ± 0.10 (3) | 0.85 ± 0.14 (3) | 133.8 ± 17.8 (4)a | 0.41 ± 0.02 (3) | 0.30 ± 0.01 (3) |
a p < 0.05 versus air value.