New mechanisms of phenytoin in calcium homeostasis: competitive inhibition of CD38 in hippocampal cells

PURPOSE: Phenytoin is a major anticonvulsant drug that is effective to improve arrhythmia and neuropathic pain. According to early works, phenytoin affected cell membrane depolarization by sodium channel blocking, guanylyl and adenylyl cyclase suppression that cause to intracellular Na(+) and Ca(2+) downregulation. This study was aimed to clarify some ambiguities in pathophysiological action of phenytoin by in vitro and molecular docking analyses. METHODS: In this study intracellular free Ca(2+) of primary culture of embryonic mouse hippocampus evaluated via Fura 2 as fluorescent probe. The effects of phenytoin on ADP ribosyl cyclase activity was assessed by recently developed fluorometric assay. Molecular docking simulation was also implemented to investigate the possible interaction between phenytoin and CD38. RESULTS: Our results confirmed phenytoin competitively inhibits cyclase activity of CD38 (IC(50) = 8.1 μM) and reduces cADPR content. cADPR is a Ca(2+)-mobilising second messenger which binds to L-type calcium channel and ryanodine receptors in cell and ER membrane and increases cytosolic free Ca(2+). Ca(2+) content of cells decreased significantly in the presence of phenytoin in a dose dependent manner (IC(50) = 12.74 µM). Based on molecular docking analysis, phenytoin binds to deeper site of CD38 active site, mainly via hydrophobic interactions and consequently inhibits proper contact of substrate with catalytic residues specially Glu 226, Trp 186, Thr221. CONCLUSION: Taken together, one of the anticonvulsant mechanisms of phenytoin is Ca(2+) inhibition from CD38 pathway, therefore could be used in disorders that accompanied by CD38 over production or activation such as heart disease, depression, brain sepsis, airway disease, oxidative stress and inflammation. [Figure: see text]