Repetitive Peroxide Exposure Reveals Pleiotropic Mitogen-Activated Protein Kinase Signaling Mechanisms
Figure 3
Chemical sensitivity and tachyphylaxis of the ERK activation response to the “acute-recovery” paradigm of hydrogen peroxide exposure. (a) Representative western blot of the chemical sensitivity of the acute-recovery paradigm peroxide-induced ERK activation (10 minute peroxide exposure, 10 minute recovery). Signaling reagents were pre-incubated with the PC12 cells prior to the “acute-recovery” peroxide exposure as follows: AG1478, 100 nM, 30 minutes; PD98059, 20 μM, 60 minutes; BAPTA-AM, 50 μM, 30 minutes; wortmannin, 10 nM, 30 minutes; PP2, 5 μM, 30 minutes; H-89, 10 μM, 30 minutes. (b) Histogram depicting the relative effects of the chemical pre-exposures from panel A to the fold over basal-induced ERK1/2 activation induced by the acute-recovery peroxide protocol. Each histogram bar depicts the mean ± standard error. (c) Diagrammatic representation of peroxide exposure procedures employed to derive protein extracts for examination of repeated peroxide exposure (grey panels) effects upon ERK1/2 activation (NS1—non-stimulated control ERK1/2 sample, R1—sample from 10 minutes after 10 minute acute-recovery peroxide process, PS2-PS3—prior-stimulation control levels before specific repeated peroxide exposure sample, R2–R4—repeated peroxide exposure protein samples). (d) The ERK1/2 activation responses to the repeated acute-recovery peroxide exposure demonstrates tachyphylaxis. The acute-recovery R1 response (10 minute exposure, 10 minute recovery) was followed by a 30 minute recovery before the same acute-recovery process was repeated, generating the R2 response. R3 and R4 responses were created in a similar manner from cells previously stimulated with R1, R2 and then subsequent R3 and R4 acute-recovery exposures. The associated histogram represents the mean ± standard error for three individual ERK1/2 R1-R4 tachyphylaxis experiments. For statistical analysis probability values indicated are *, **.
