FIGURE 4.

Induction of ER stress with thapsigargin induces ceramide accumulation in INS-1 cells, and this is prevented by pharmacologic inhibition of p38 MAPK. INS-1 insulinoma cells stably transfected with a construct that causes overexpression of iPLA₂β (OE) or with empty vector (V) were preincubated (1 h, 37 °C) with the p38 MAPK inhibitor PD169316 (5 μm) or with vehicle diluent alone and then placed in fresh KRB medium without or with thapsigargin (1 μm). At the end of the incubation period, cells were collected, homogenized, and extracted by a modified Bligh-Dyer method. The extract was admixed with internal standard C8-ceramide, concentrated, reconstituted, infused in a solution to which Li⁺ had been added, and analyzed by ESI/MS/MS scanning for constant neutral loss (CNL) of 48 from [M + Li]⁺ ions as described under “Experimental Procedures.” Quantitation of each ceramide species was achieved by dividing the ion current at the m/z value for that species by the ion current of the internal standard at m/z 432.5 and interpolating from a calibration curve. This value was then normalized to the measured lipid phosphorus content of the sample. A and B, representative CNL spectra from INS-1 cells incubated only with vehicle or with 1 μm thapsigargin, respectively. In C and D, mean values (n = 3) for the normalized total amount of ceramide species under the indicated conditions are displayed for INS-1 cells that are transfected with empty vector (C) or that stably overexpress iPLA₂β (D), and S.E. values (error bars) are indicated. *, p < 0.05 for the comparison of the indicated condition versus the analogous condition in which incubation was performed in the presence of PD169316.