Further experiments to establish the contribution of the P2X7 receptor in cytokine-mediated MV release were carried out by the use of specific P2X7 antagonists, oxATP (29) and BBG (30). Both inhibitors were effective in reducing the exATP-triggered release of MVs (p = 0.0006 and p = 0.0002, respectively) but did not alter the cytokine-mediated release (Figures 5A–C). As some of the P2X7 functions can be ascribed to the opening of the associated hemichannel pannexin-1, the effect on MV release of two of its selective inhibitors, probenecid (31) and the small peptide 10Panx1 (32), was checked. Also in this case, no reduction was observed in the number of MVs released by cells treated with cytokines in the presence of the pannexin-1 inhibitors (Figures 5D–F). According to a previous study (7), the activation of the lipid-metabolizing enzyme acid sphingomyelinase (ASMase) is a crucial step for the formation of MVs due to the generation of ceramide and phosphorylcoline from sphingomyelin. We chose imipramine and siramesine, two structurally unrelated ASMase antagonists (33), to verify the contribution of this enzyme on MV release mediated by IFN-γ and IL-4. The two drugs were not effective in modulating such process (Figures 5G–I). Overall, this result suggests that, in order to induce the release of MVs, ATP, and cytokines operate along very different pathways, not only in terms of signaling activation but also, more interestingly, on the basic biochemical/structural processes underlying MV formation at the plasma membrane. Additionally, considering that (i) the kinetics of the two processes are so different in time (minutes versus several hours) and (ii) many effects of cytokines are mediated by activation of transcriptional factors, we sought to determine whether inhibition of transcription could distinguish the two systems.