TABLE 1.
Modulation of [3H]arachidonic acid release and [3H]GroPIns production induced by FcR-mediated phagocytosis and LPS stimulation in Raw264.7 cells
Raw264.7 cells without or with 15-min preincubations with the different inhibitors, as indicated, were left untreated or were stimulated by FcR-mediated phagocytosis or with 20 μg/ml LPS for a further 30 and 45 min, respectively, at 37 °C (see “Experimental Procedures”). [3H]Arachidonic acid release as percentage of total [3H]arachidonic acid cell content, with basal release before phagocytosis and LPS stimulation of 0.93 ± 0.06 and 1.12 ± 0.07% of total cell content, respectively. Intracellular [3H]GroPIns production as a percentage of total water-soluble [3H]inositol-labeled metabolites, with the basal levels before phagocytosis and LPS stimulation of 9.98% ± 0.60 and 8.12% ± 0.62%, respectively. Data are means ± S.E. of at least four independent experiments, each performed in duplicate. In parallel, under the conditions reported above, lysophosphatidylinositol levels were monitored by TLC (for the method see Ref. 42), with an increase of 0.3- and 0.8-fold over basal for stimulation by FcR-mediated phagocytosis and LPS, respectively, effects that were completely blocked by pyrrophenone treatment. ND means not determined.
| Condition (target) | [3H]arachidonic acid release (% total cell content) |
[3H]GroPIns production (% total water-soluble [3H]inositol cell content) |
||||
|---|---|---|---|---|---|---|
| Unstimulated | FcR-phagocytosis | LPS | Unstimulated | FcR-phagocytosis | LPS | |
| Control | 1.93 ± 0.17a | 1.95 ± 0.18a | 14.88 ± 0.61b | 18.03 ± 2.56b | ||
| 0.5 μm Pyr (PLA2IVα) | 0.98 ± 0.04 | 0.97 ± 0.08c | 0.97 ± 0.08a | 5.44 ± 0.79 | 8.04 ± 0.81b | 6.34 ± 0.85b |
| 1 μm BEL (PLA2VI) | 1.03 ± 0.06 | 2.00 ± 0.08 | 1.55 ± 0.10 | 6.42 ± 0.41 | 16.33 ± 0.01 | 19.91 ± 0.81 |
| 50 μm sPLA2IIA inhibitor-I (sPLA2) | 1.16 ± 0.07 | 2.10 ± 0.04 | 1.69 ± 0.27 | 10.31 ± 1.05 | 21.14 ± 0.01 | 20.54 ± 2.76 |
| 10 μm SB203580 (p38) | 1.30 ± 0.11 | 1.35 ± 0.06c | 1.83 ± 0.32 | 6.08 ± 0.62 | ND | 13.49 ± 2.56 |
| 10 μm U0126 (ERK1/2) | 1.16 ± 0.28 | 1.25 ± 0.04c | 1.08 ± 0.17b | 5.81 ± 0.62 | ND | 6.39 ± 0.62b |
| 10 μm SP600125 (JNK) | 1.16 ± 0.04 | 1.20 ± 0.03a | 2.61 ± 0.04 | ND | ND | ND |
| 10 μm SB203580 + 10 μm U0126 (p38 + ERK1/2) | ND | 0.87 ± 0.15c | ND | ND | ND | ND |
a p < 0.001 (Student's t test), with respect to their relevant controls.
b p < 0.05 (Student's t test), with respect to their relevant controls.
c p < 0.02 (Student's t test), with respect to their relevant controls.