In order to address whether activation of the NOX/XOX pathway by EtOH/Ach resulted in ROS generation, we analyzed the changes in NOX/XOX protein contents in cell lysates derived from neuronal cultures after 48 h EtOH/Ach exposure in the presence or absence of cyclohexamide (Chx; protein synthesis inhibitor) or actinomycin D (ACD; inhibitor of RNA synthesis). Western blot analyses revealed that EtOH or Ach respectively increased the NOX protein contents by 34 or 43%, while XOX levels were elevated by 16 or 20% compared with respective controls (Figs. 4A-D). Pretreatment of neurons with ACD effectively diminished the Ach-induced increase in NOX (43%) or XOX protein levels (20%) to the basal level, while Chx had minimal effects on NOX (P = 0.041), suggesting that EtOH/Ach modulated the transcription of NOX/XOX mRNA level rather than translation. To confirm this observation, we examined the changes in NOX/XOX mRNA levels by real-time PCR in RNA extracts from primary neurons treated with EtOH/Ach in the presence or absence of Chx or ACD for 48 h. EtOH/Ach exposure increased the NOX mRNA levels by 47 or 51%,
