FIGURE 7.

PKC and PI3K are important to ROS generation induced by heme. Peritoneal macrophages from C57Bl/6 were treated with 10 μm of the PKC inhibitor calphostin (A) or 50 μm of the PI3K inhibitor wortmannin (B) for 30 min and stimulated with heme (100 μm) for 1 h. The ROS generation was evaluated by flow cytometry using the probe CM-H2DCFDA (2 μm). Inhibitors plus heme (thick line), heme (filled area curve), probe (fine line). The results are representative of three different experiments. C, macrophages were pretreated for 30 min with calphostin (10 μm) or wortmannin (50 μm) and were stimulated with heme, LPS, or heme plus LPS. TNF was quantified by ELISA in supernatants collected 4 h after treatment with heme and LPS. All experiments were performed with medium supplemented with 10% FCS. Results represent mean ± S.E. and are representative of three different experiments. *, p < 0.05.