DARU Journal of Pharmaceutical Sciences

, Volume 26, Issue 2, pp 191–198 | Cite as

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

  • Leila SadeghiEmail author
  • Reza Yekta
  • Gholamreza Dehghan
Research Article



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 Ca2+ downregulation. This study was aimed to clarify some ambiguities in pathophysiological action of phenytoin by in vitro and molecular docking analyses.


In this study intracellular free Ca2+ 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.


Our results confirmed phenytoin competitively inhibits cyclase activity of CD38 (IC50 = 8.1 μM) and reduces cADPR content. cADPR is a Ca2+-mobilising second messenger which binds to L-type calcium channel and ryanodine receptors in cell and ER membrane and increases cytosolic free Ca2+. Ca2+ content of cells decreased significantly in the presence of phenytoin in a dose dependent manner (IC50 = 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.


Taken together, one of the anticonvulsant mechanisms of phenytoin is Ca2+ 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.

Graphical abstract


Phenytoin CD38 Competitive inhibition Calcium homeostasis Sodium blocker Membrane depolarization 


Compliance with ethical standards

Conflict of interest

All of the Authors have no conflict of interest to declare.


  1. 1.
    Gallop K. Review article: phenytoin use and efficacy in the ED. Emerg Med Australas. 2010;22(2):108–18.PubMedGoogle Scholar
  2. 2.
    Jones GL, Wimbish GH, McIntosh WE. Phenytoin: basic and clinical pharmacology. Med Res Rev. 1983;3(4):383–434.CrossRefPubMedGoogle Scholar
  3. 3.
    Guldiken B, Remi J, Noachtar H. Cardiovascular adverse effects of phenytoin. J Neurol. 2016;263(5):861–70.CrossRefPubMedGoogle Scholar
  4. 4.
    Jensen TS. Anticonvulsants in neuropathic pain: rationale and clinical evidence. Eur J Pain. 2002;6:61–8.CrossRefPubMedGoogle Scholar
  5. 5.
    Nelson M, Yang M, Dowle AA, Thomas JR, Brackenbury WJ. The sodium channel-blocking antiepileptic drug phenytoin inhibits breast tumour growth and metastasis. Mol Cancer. 2015;27(14):13.CrossRefGoogle Scholar
  6. 6.
    Abdelsayed M, Sokolov S. Voltage gated sodium channels: pharmaceutical targets via anticonvulsants to treat epilepticsyndromes. Channels (Austin). 2013;7(3):146–52.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Backus KH, Pflimlin P, Trube G. Action of diazepam on the voltage-dependent Na+ current. Comparison with the effects of phenytoin, carbamazepine, lidocaine and flumazenil. Brain Res. 1991;548(1–2):41–9.CrossRefPubMedGoogle Scholar
  8. 8.
    Ferrendelli JA, Kinscherf DA. Phenytoin: effects on calcium flux and cyclic nucleotides. Epilepsia. 1977;18(3):331–6.CrossRefPubMedGoogle Scholar
  9. 9.
    Twombly DA, Yoshii M, Narahashi T. Mechanisms of calcium channel block by phenytoin. J Pharmacol Exp Ther. 1988;246(1):189–95.PubMedGoogle Scholar
  10. 10.
    Khaira A, Gupta A, Madhu SV, Khaira DD. Phenytoin induced severe disabling osteomalacia in a young male with seizure disorder. J Assoc Physicians India. 2008;56:376–8.PubMedGoogle Scholar
  11. 11.
    Bruzzone S, Moreschi I, Guida L, Usai C, Zocchi E, De Flora A. Extracellular NAD+ regulates intracellular calcium levels and induces activation of humangranulocytes. Biochem J. 2006;393(3):697–704.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Liu Q, Kriksunov IA, Graeff R, Munshi C, Lee HC, Hao Q. Structural basi for the mechanistic understanding of human CD38-controlled multiple catalysis. J Biol Chem. 2006;281(43):32861–9.CrossRefPubMedGoogle Scholar
  13. 13.
    Partida-Sánchez S, Iribarren P, Moreno-García ME, Gao JL, Murphy PM, Oppenheimer N, et al. Chemotaxis and calcium responses of phagocytes to formyl peptide receptor ligands is differentially regulated by cyclic ADP ribose. J Immunol. 2004;172(3):1896–906.CrossRefPubMedGoogle Scholar
  14. 14.
    Zündorf G, Reiser G. Calcium dysregulation and homeostasis of neural calcium in the molecular mechanisms of neurodegenerative diseases provide multiple targets for neuroprotection. Antioxid Redox Signal. 2011;14(7):1275–88.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Seibenhener ML, Wooten MW. Isolation and culture of hippocampal neurons from prenatal mice. J Vis Exp. 2012;65(3634):1–6.Google Scholar
  16. 16.
    Amina S, Hashii M, Ma WJ, Yokoyama S, Lopatina O, Liu HX, et al. Intracellular calcium elevation induced by extracellular application of cyclic-ADP-ribose or oxytocin is temperature-sensitive in rodent NG108-15 neuronal cells with or without exogenous expression of human oxytocin receptors. J Neuroendocrinol. 2010;22:460–6.CrossRefPubMedGoogle Scholar
  17. 17.
    Brewer GJ. Serum-free B27/neurobasal medium supports differentiated growth of neurons from the striatum, substantia nigra, septum, cerebral cortex, cerebellum, and dentate gyrus. J Neurosci Res. 1995;42:674–83.CrossRefPubMedGoogle Scholar
  18. 18.
    Hirst RA, Harrison C, Hirota K, Lambert DG. Measurement of [Ca2+]i in whole cell suspensions using fura-2. Methods Mol Biol. 2005;312:37–45.PubMedGoogle Scholar
  19. 19.
    Bagal SK, Marron BE, Owen RM, Storer RI, Swain NA. Voltage gated sodium channels as drug discovery targets. Channels (Austin). 2015;9(6):360–6.CrossRefGoogle Scholar
  20. 20.
    Sun L, Adebanjo OA, Moonga BS, Corisdeo S, Anandatheerthavarada HK, Biswas G, et al. CD38/ADP-ribosyl cyclase: a new role in the regulation of osteoclastic bone resorption. J Cell Biol. 1999;146(5):1161–72.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, et al. AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J Comput Chem. 2009;30:2785–91.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Rizvi SMD, Shakil S. Haneef M. a simple click by click protocol to perform docking: AutoDock 4.2 made easy for non-bioinformaticians. EXCLI J. 2013;12:831–57.PubMedPubMedCentralGoogle Scholar
  23. 23.
    Graeff RM, Mehta K, Lee HC. GDP-ribosyl cyclase activity as a measure of CD38 induction by retinoic acid in HL-60 cells. Biochem Biophys Res Commun. 1994;205(1):722–7.CrossRefPubMedGoogle Scholar
  24. 24.
    Liu Q, Graeff R, Kriksunov IA, Jiang H, Zhang B, Oppenheimer N, et al. Structural basis for enzymatic evolution from a dedicated ADP-ribosyl cyclase to a multifunctional NAD hydrolase. J Biol Chem. 2009;284:27637–45.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Liu Q, Kriksunov IA, Graeff R, Munshi C, Lee HC, Hao Q. Structural basis for the mechanistic understanding of human CD38-controlled multiple catalysis. J Biol Chem. 2006;281:32861–9.CrossRefPubMedGoogle Scholar
  26. 26.
    Meyer EA, Castellano RK, Diederich F. Interactions with aromatic rings in chemical and biological recognition. Angew Chem Int Ed Engl. 2003;42(11):1210–50.CrossRefPubMedGoogle Scholar
  27. 27.
    Lucas X, Bauzá A, Frontera A, Quiñonero D. A thorough anion-π interaction study in biomolecules: on the importance of cooperativity effects. Chem Sci. 2016;7(2):1038–50.CrossRefPubMedGoogle Scholar
  28. 28.
    Zhang H, Graeff R, Lee HC, Hao Q. Crystal structures of human CD38 in complex with NAADP and ADPRP. Messenger. 2013;2(1):44–53.CrossRefGoogle Scholar
  29. 29.
    Graeff R, Liu Q, Kriksunov IA, Hao Q, Lee HC. Acidic residues at the active sites of CD38 and ADP-ribosyl cyclase determine nicotinic acid adenine dinucleotide phosphate (NAADP) synthesis and hydrolysis activities. J Biol Chem. 2006;281(39):28951–7.CrossRefPubMedGoogle Scholar
  30. 30.
    Peng QY, Wang YM, Chen CX, Zou Y, Zhang LN, Deng SY, et al. Inhibiting the CD38/cADPR pathway protected rats against sepsis associated brain injury. Brain Res. 2018;1678:56–63.CrossRefPubMedGoogle Scholar
  31. 31.
    Antonelli A, Ferrannini E. CD38 autoimmunity: recent advances and relevance to human diabetes. J Endocrinol Investig. 2004;27(7):695–707.CrossRefGoogle Scholar
  32. 32.
    Thornton PD, Fernandez C, Giustolisi GM, Morilla R, Atkinson S, A’Hern RP, et al. CD38 expression as a prognostic indicator in chronic lymphocytic leukaemia. Hemato J. 2004;5:145–51.CrossRefGoogle Scholar
  33. 33.
    Roussanov BV, Taylor JM, Giorgi JV. Calculation and use of an HIV-1 disease progression score. AIDS. 2000;14(17):2715–22.CrossRefPubMedGoogle Scholar
  34. 34.
    Gul R, Park JH, Kim SY, Jang KY, Chae JK, Ko JK, et al. Inhibition of ADP-ribosyl cyclase attenuates angiotensin II induced cardiac hypertrophy. Cardiovasc Res. 2009;81(3):582–91.CrossRefPubMedGoogle Scholar
  35. 35.
    Moreau C, Liu Q, Graeff R, Wagner GK, Thomas MP, Swarbrick JM, et al. CD38 Structure-Based Inhibitor Design Using the N1-Cyclic Inosine 59-Diphosphate Ribose Template. PLoS One. 2013;8(6):66247.CrossRefGoogle Scholar
  36. 36.
    Bose T, Cieślar-Pobuda A, Wiechec E. Role of ion channels in regulating Ca2+ homeostasis during the interplay between immune and cancer cells. Cell Death Dis. 2015;6:e1648.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Wei W, Graeff R, Yue J. Roles and mechanisms of the CD38/cyclic adenosine diphosphate ribose/Ca2+ signaling pathway. World J Biol Chem. 2014;5(1):58–67.CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Lee KH, Park JY, Kim K. NMDA receptor-mediated calcium influx plays an essential role in myoblast fusion. Volume 578, Issues 1–2, 3 December 2004, Pages 47–52.Google Scholar
  39. 39.
    Zhang Y, Chen X, Gueydan C, Han J. Plasma membrane changes during programmed cell deaths. Cell Res. 2018;28(1):9–21.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2018

Authors and Affiliations

  1. 1.Department of Animal Biology, Faculty of Natural SciencesUniversity of TabrizTabrizIran

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