Role of cytochrome P₄₅₀ (CYP) and nuclear factor (NF)- κB in D-meth-induced ceramide production.

<p>(A, B) Effects of pan-CYP inhibitor clotrimazole (CLO) on (A) cell-associated D-meth content and (B) ceramide levels. Primary MEF cultures were treated with D-meth (M, 1 mM) for 24 h and rinsed before extraction and quantification of D-meth by LC/MS. (C) Effects of CYP inhibitors on ceramide levels: SKF-525A (SKF, 10 μM), cimetidine (CIM, 10 μM), quinidine (QUI, 10 μM) and HET-0016 (HET, 10 μM). (D-E) Time-course of the effects of (D) D-meth (mM) and (E) 4-hydroxy-D-methamphetamine (4-OH, 1 mM) on ROS production. (F-G) Effects of (F) clotrimazole and (G) SKF-525A, cimetidine, quinidine and HET-0016 on ROS production. (H) Chromatin immunoprecipitation indicating recruitment of NF-κB to the TNF- promoter in primary MEF treated for 24 h with vehicle or D-meth (1 mM). Results are shown as percent of input (i.e., percent of the total amount of chromatin before immunoprecipitation). (I-K) Effects of NF-κB inhibitors (I) thalidomide (THA, 25 M), (J) 5-aminosalicylic acid (ASA, 10 μM) and (K) JSH-23 (JSH, 10 μM) on ceramide levels in MEF treated for 24h with vehicle or D-meth (1 mM). Values are expressed as mean±s.e.m. of three separate experiments, each performed in triplicate. *P<0.05, ***P<0.001, ANOVA followed by Bonferroni post hoc test.