TABLE 1.
Impact of various agents on .NO-mediated inhibition of β2 integrin-dependent murine neutrophil adherence, iNOS activation, DCF-DA fluorescence, and actin polymerization
Neutrophils were exposed to PBS or 100 nm DENO for 2 min before assays were performed. Cell adherence reflects β2 integrin-specific adherence to fibrinogen-coated plates. NOS activity reflects pmol [H3]citrulline produced when [H3]arginine was added to cells that were then incubated for 15 min. DCF fluorescence was assessed when 10 μm DCF-DA was added to cell suspensions and monitored for up to 10 min. Actin polymerization reflect pyrene actin fluorescence in arbitrary units/min. All values are means ± S.E. (n = number of independent trials). Where studies were done with small chemical inhibitors, cells were incubated with the agent for 10 min prior to adding DENO or an equal volume of PBS (control) before performing the assay. Where studies were done with siRNA, cells were incubated with control, scrambled sequence, or siRNA sequences specific for the protein indicated for 24 h prior to performing the study. iNOS KO, neutrophils from NOS-2 knock-out mice; gp91phox KO, neutrophils from knock-out mice lacking the gp91phox subunit of NADPH oxidase; 1400W, incubation with 1 mm 1400W; UV, cells exposed to UV light for 5 min after PBS or DENO incubation for 2 min; Cyto D, incubation with 5 μm cytochalasin D; Nox2ds, incubation with 10 μm Nox-2ds; Scrmb-Nox2ds, incubations performed with 10 μm control, scrambled sequence peptide to Nox-2ds; Rac-i, incubation with 50 μm NSC 23766; Cont-si, cells incubated with control scrambled sequence siRNA; gp91phox-si, cells incubated with siRNA to gp91phox; VASP-si, cells incubated with siRNA to vasodilator stimulated phosphoprotein; FAK-i, cells incubated with 20 μm PT 573228; FAK-si, cells incubated siRNA to focal adhesion kinase; Geld, cells incubated with 3 μm geldanamycin; HSP90-si, cells incubated with siRNA to HSP90; PDI-i, cells incubated with 30 μm quercetin-3-rutinoside, PDI-si, cells incubated with siRNA to protein disulfide isomerase; mGC-si, cells incubated with siRNA to membrane guanylate cyclase; ND, no experiments performed.
| Agent | Neutrophil adherence |
iNOS |
DCF |
Actin polymerization |
||||
|---|---|---|---|---|---|---|---|---|
| PBS | DENO | PBS | DENO | PBS | DENO | PBS | DENO | |
| % | pmol [H3] citrulline/h | fluorescence/min | fluor/min × 103 | |||||
| PBS | 20.6 ± 0.4 (21) | 2.4 ± 0.4 (20)a | 0.20 ± 0.05 (15) | 2.38 ± 0.05 (12)a | 0.88 ± 0.09 (16) | 2.2 ± 0.10 (7)a | 1.00 ± 0.06 (8) | 3.63 ± 0.26 (8)a |
| iNOS KO | 19.7 ± 0.2 (5) | 18.3 ± 0.3 (5) | 0 ± 0 (5) | 0 ± 0 (5) | ND | ND | 0.80 ± 0.20 (3) | 1.10 ± 0.06 (6) |
| gp91phox KO | 20.4 ± 0.8 (3) | 20.0 ± 0.7 (4) | ND | ND | ND | ND | 0.93 ± 0.23 (3) | 1.00 ± 0.12 (4) |
| 1400W | 21.3 ± 0.5 (5) | 20.8 ± 0.7 (5) | 0 ± 0 (8) | 0 ± 0 (8) | 0.98 ± 0.07 (3) | 1.08 ± 0.11 (3) | 0.87 ± 0.13 (5) | 1.00 ± 0.02 (3) |
| Nox2ds | 20.1 ± 0.1 (5) | 19.7 ± 0.5 (5) | 0.25 ± 0.05 (6) | 0.32 ± 0.05 (4) | 1.00 ± 0.01 (3) | 0.67 ± 0.33 (3) | 1.05 ± 0.13 (4) | 0.76 ± 0.11 (5) |
| Scrmb- Nox2ds | 22.2 ± 0.9 (4) | 3.4 ± 0.8 (4)a | 0.21 ± 0.09 (3) | 2.0 ± 0.18 (4)a | 1.17 ± 0.17 (3) | 1.99 ± 0.03 (3)a | 1.1 ± 0.15 (7) | 3.11 ± 0.25 (3)a |
| Cont-si | 17.1 ± 1.2 (12) | 2.7 ± 0.4 (10)a | 0.20 ± 0.02 (9) | 2.1 ± 0.16 (7)a | 1.10 ± 0.16 (9) | 2.51 ± 0.22 (8)a | 0.94 ± 0.11 (5) | 4.04 ± 0.44 (5)a |
| gp91phox-si | 20.6 ± 0.9 (3) | 20.3 ± 0.1 (3) | 0.23 ± 0.13 (3) | 0.40 ± 0.13 (3) | 0.98 ± 0.33 (3) | 1.00 ± 0.27 (3) | ND | ND |
| PDI-i | 22.6 ± 0.2 (3) | 20.8 ± 0.9 (3) | 0.21 ± 0.08 (3) | 0. 61 ± 0.06 (3) | 1.00 ± 0.08 (3) | 0.67 ± 0.14 (3) | 0.53 ± 0.24 (3) | 0.57 ± 0.07 (3) |
| PDI-si | 22.6 ± 0.3 (3) | 20.3 ± 0.2 (3) | 0.21 ± 0.10 (6) | 0.33 ± 0.10 (6) | 1.00 ± 0.33 (3) | 1.00 ± 0.38 (3) | 1.00 ± 0.03 (3) | 1.03 ± 0.03 (3) |
| UV | 21.4 ± 0.6 (6) | 20.7 ± 0.7 (6) | 0.13 ± 0.03 (7) | 0.25 ± 0.01 (4) | 1.00 ± 0.11 (3) | 1.1 ± 0.10 (3) | 0.90 ± 0.10 (3) | 0.90 ± 0.08 (6) |
| Cyto D | 22.4 ± 0.4 (3) | 21.1 ± 0.3 (3) | 0.15 ± 0.04 (6) | 0.20 ± 0.08 (3) | 0.98 ± 0.17 (3) | 1.1 ± 0.17 (3) | 0.78 ± 0.13 (4) | 0.85 ± 0.13 (6) |
| Rac-i | 20.7 ± 1.1 (5) | 19.5 ± 0.7 (6) | 0.21 ± 0.01 (5) | 0.32 ± 0.04 (4) | 1.05 ± 0.07 (3) | 0.70 ± 0.02 (3) | 0.87 ± 0.08 (3) | 0.85 ± 0.13 (6) |
| VASP-si | 14.5 ± 1.2 (3) | 14.7 ± 0.9 (5) | 0.20 ± 0.03 (4) | 0.19 ± 0.01 (4) | 0.80 ± 0.20 (3) | 0.22 ± 0.15 (3)a | 1.10 ± 0.10 (3) | 0.90 ± 0.10 (3) |
| FAK-i | 21.6 ± 1.5 (3) | 20.6 ± 1.3 (3) | 0.23 ± 0.03 (3) | 0.35 ± 0.06 (3) | 1.00 ± 0.08 (3) | 0.28 ± 0.03 (3) | 0.97 ± 0.03 (3) | 0.10 ± 0.01 (3) |
| FAK-si | 15.8 ± 1.7 (3) | 14.1 ± 1.9 (4) | 0.19 ± 0.01 (4) | 0.20 ± 0.02 (4) | 0.92 ± 0.08 (3) | 0.27 ± 0.03 (3) | 1.10 ± 0.09 (3) | 1.12 ± 0.10 (3) |
| Geld | 21.1 ± 1.2 (5) | 20.7 ± 0.6 (5) | 0.31 ± 0.0 (3) | 0.39 ± 0.05 (3) | 0.83 ± 0.17 (3) | 1.10 ± 0.33 (3) | 1.17 ± 0.17 (5) | 1.23 ± 0.15 (3) |
| HSP90-si | 20.1 ± 0.9 (5) | 18.7 ± 0.7 (5) | 0.24 ± 0.02 (3) | 0.39 ± 0.08 (3) | 0.95 ± 0.23 (6) | 0.97 ± 0.19 (6) | 1.10 ± 0.07 (3) | 1.33 ± 0.17 (3) |
| 8-br-cGMP | 20.7 ± 0.2 (4) | 20.3 ± 0.1 (4) | 0.24 ± 0.04 (3) | 0.32 ± 0.07 (3) | 1.10 ± 0.10 (3) | 1.11 ± 0.08 (3) | 1.10 ± 0.10 (3) | 1.17 ± 0.09 (3) |
| mGC-si | 21.8 ± 0.7 (5) | 20.0 ± 0.2 (5) | 0.23 ± 0.07 (5) | 0.33 ± 0.10 (5) | 0.68 ± 0.10 (3) | 0.67 ± 0.08 (3) | 1.00 ± 0.01 (3) | 1.01 ± 0.03 (3) |
a p < 0.05 versus control/PBS value