FIGURE 7.

Morphine withdrawal hyperactivates ERK1/2 with corticosterone treatment in LPS-stimulated CRL2019 cells, and ERK1/2 activation is inhibited by PD98059. CRL2019 cells were treated with PD98059 for 1 h before treating with 100 ng/ml LPS (6 h) and/or 300 ng/ml corticosterone (30 min) before the end of LPS treatment following the morphine withdrawal paradigm as discussed under “Experimental Procedures.” Total cell extract was prepared, and 50 μg of total proteins were loaded onto 10% denaturing gel. A, the membranes were probed with phosphorylated isoforms and total isoforms of p38 and SAPK/JNK MAPKs. The blots were reprobed with α-tubulin as a loading control. B, the membranes were probed with phosphorylated isoforms and total isoforms of ERK1/2 MAPK antibodies. The blots were reprobed with α-tubulin as a loading control. C, cells were treated with PD98059 (40 μm) for 1 h before treating with LPS (100 ng/ml) for 0, 24, 48, and 72 h following morphine withdrawal. Corticosterone treatment was done for 30 min at the end of LPS treatment, and the cultured medium was collected to perform ELISA for IL-12p40. IL-12p40 protein was expressed as pg/ml concentration for LPS alone (□), LPS with morphine withdrawal (×), LPS with corticosterone (CS) (△), LPS with corticosterone in MW samples (○), or LPS plus corticosterone with MW in the presence of PD98059 (PD) (♢). Each experiment is a representation of at least three independent experiment ± S.D. (error bars). *, p < 0.05; **, p < 0.01 compared with LPS alone samples.