Skip to main content

Inhibitions of anandamide transport and FAAH synthesis decrease apoptosis and oxidative stress through inhibition of TRPV1 channel in an in vitro seizure model

Molecular and Cellular Biochemistry volume 453, pages 143–155 (2019)Cite this article

Abstract

The expression level of TRPV1 is high in hippocampus which is a main epileptic area in the brain. In addition to the actions of capsaicin (CAP) and reactive oxygen species (ROS), the TRPV1 channel is activated in neurons by endogenous cannabinoid, anandamide (AEA). In the current study, we investigated the role of inhibitors of TRPV1 (capsazepine, CPZ), AEA transport (AM404), and FAAH (URB597) on the modulation of Ca2+ entry, apoptosis, and oxidative stress in in vitro seizure-induced rat hippocampus and human glioblastoma (DBTRG) cell line. The seizure was induced in the hippocampal and DBTRG neurons using in vitro 4-aminopyridine (4-AP) to trigger a seizure-like activity model. CPZ and AM404 were fully effective in reversing 4-AP-induced intracellular free Ca2+ concentration of the hippocampus and TRPV1 current density of DBTRG. However, AEA and CAP did not activate TRPV1 in the URB597-treated neurons. Hence, we observed TRPV1 blocker effects of URB597 in the DBTRG neurons. In addition, the AM404 and CPZ treatments decreased intracellular ROS production, mitochondrial membrane depolarization, apoptosis, caspases 3 and 9 values in the hippocampus. In conclusion, the results indicate that inhibition of AEA transport, FAAH synthesis, and TRPV1 activity can result in remarkable neuroprotective effects in the epileptic neurons.

Graphical abstract

Possible molecular pathways of involvement of capsazepine (CPZ) and AM4040 in anandamide and capsaicin (CAP)-induced apoptosis, oxidative stress, and Ca2+ accumulation through TRPV1 channel in the seizure-induced rat hippocampus and human glioblastoma neurons. The TRPV1 channel is activated by different stimuli including reactive oxygen species (ROS), anandamide (AEA), and CAP and it is blocked by capsazepine (CPZ). Cannabinoid receptor type 1 (CB1) is also activated by AEA. The AEA levels in cytosol are decreased by fatty acid amide hydrolase (FAAH) enzyme. Inhibition of FAAH through URB597 induces stimulation of CB1 receptor through accumulation AEA. URB597 acts antiepileptic effects through inhibition of TRPV1. Overloaded Ca2+ concentration of mitochondria can induce an apoptotic program by stimulating the release of apoptosis-promoting factors such as caspases 3 and caspase 9 by generating ROS due to respiratory chain damage. AM404 and CPZ reduce TRPV1 channel activation and Ca2+ entry in the in vitro 4-AP seizure model-induced hippocampal and glioblastoma neurons.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

USD 39.95

Price includes VAT (USA)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Abbreviations

[Ca2+]i :

Intracellular free calcium ion

4-AP:

4-Aminopyridine

aCSF:

Artificial cerebrospinal fluid

AEA:

Anandamide

CAP:

Capsaicin

CPZ:

Capsazepine

CRR:

Cannabinoid reuptake

DBTRG:

Denver Brain Tumor Research Group

EGTA:

Ethylene glycol-bis[2-aminoethyl-ether]-N,N,N,N-tetraacetic acid

FAAH:

Fatty acid amide hydrolase

HBSS:

Hank’s buffered salt solution

IRTX:

5′-Iodoresiniferatoxin

RNS:

Reactive nitrogen species

ROS:

Reactive oxygen species

TRP:

Transient receptor potential

TRP:

Transient receptor potential

TRPV1:

Transient receptor potential vanilloid 1

VGCC:

Voltage-gated calcium channels

References

  1. Iannotti FA, Hill CL, Leo A et al (2014) Nonpsychotropic plant cannabinoids, cannabidivarin (CBDV) and cannabidiol (CBD), activate and desensitize transient receptor potential vanilloid 1 (TRPV1) channels in vitro: Potential for the treatment of neuronal hyperexcitability. ACS Chem Neurosci 5:1131–1141

    Article  CAS  PubMed  Google Scholar 

  2. Gonzalez-Reyes LE, Ladas TP, Chiang CC, Durand DM (2013) TRPV1 antagonist capsazepine suppresses 4-AP-induced epileptiform activity in vitro and electrographic seizures in vivo. Exp Neurol 250:321–332

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Manjarrez-Marmolejo J, Franco-Pérez J (2016) Gap junction blockers: an overview of their effects on induced seizures in animal models. Curr Neuropharmacol 14:759–771

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Bhaskaran MD, Smith BN (2010) Effects of TRPV1 activation on synaptic excitation in the dentate gyrus of a mouse model of temporal lobe epilepsy. Exp Neurol 223:529–536

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. von Rüden EL, Jafari M, Bogdanovic RM, Wotjak CT, Potschka H (2014) Analysis in conditional cannabinoid 1 receptor-knockout mice reveals neuronal subpopulation-specific effects on epileptogenesis in the kindling paradigm. Neurobiol Dis 37:334–347

    Google Scholar 

  6. Nazıroğlu M, Övey İS (2015) Involvement of apoptosis and calcium accumulation through TRPV1 channels in neurobiology of epilepsy. Neuroscience 293:55–66

    Article  CAS  PubMed  Google Scholar 

  7. Nazıroğlu M, Yürekli VA (2013) Effects of antiepileptic drugs on antioxidant and oxidant molecular pathways: focus on trace elements. Cell Mol Neurosci 33:589–599

    Article  CAS  Google Scholar 

  8. Carrasco C, Naziroǧlu M, Rodríguez AB, Pariente JA (2018) Neuropathic pain: delving into the oxidative origin and the possible implication of transient receptor potential channels. Front Physiol 9:95

    Article  PubMed  PubMed Central  Google Scholar 

  9. Ogawa N, Kurokawa T, Mori Y (2016) Sensing of redox status by TRP channels. Cell Calcium 60(2):115–122

    Article  CAS  PubMed  Google Scholar 

  10. Nazıroğlu M, Çiğ B, Blum W et al (2017) Targeting breast cancer cells by MRS1477, a positive allosteric modulator of TRPV1 channels. PLoS ONE 12:e0179950

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Nazıroğlu M (2015) TRPV1 channel: a potential drug target for treating epilepsy. Curr Neuropharmacol 13:239–247

    Article  PubMed  PubMed Central  Google Scholar 

  12. Cristino L, de Petrocellis L, Pryce G, Baker D, Guglielmotti V, Di Marzo V (2006) Immunohistochemical localization of cannabinoid type 1 and vanilloid transient receptor potential vanilloid type 1 receptors in the mouse brain. Neuroscience 139:1405–1415

    Article  CAS  PubMed  Google Scholar 

  13. Al-Hayani A, Wease KN, Ross RA, Pertwee RG, Davies SN (2001) The endogenous cannabinoid anandamide activates vanilloid receptors in the rat hippocampal slice. Neuropharmacology 41:1000–1005

    Article  CAS  PubMed  Google Scholar 

  14. Gobira PH, Lima IV, Batista LA et al (2017) N-Arachidonoyl-serotonin, a dual FAAH and TRPV1 blocker, inhibits the retrieval of contextual fear memory: role of the cannabinoid CB1 receptor in the dorsal hippocampus. J Psychopharmacol 31(6):750–756

    Article  CAS  PubMed  Google Scholar 

  15. Beltramo M, de Fonseca FR, Navarro M et al (2000) Reversal of dopamine D(2) receptor responses by an anandamide transport inhibitor. J Neurosci 20:3401–3407

    Article  CAS  PubMed  Google Scholar 

  16. Devane WA, Hanus L, Breuer A et al (1992) Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science 258:1946–1949

    Article  CAS  PubMed  Google Scholar 

  17. Mikheeva IB, Shubina L, Matveeva N, Pavlik LL, Kitchigina VF (2017) Fatty acid amide hydrolase inhibitor URB597 may protect against kainic acid-induced damage to hippocampal neurons: dependence on the degree of injury. Epilepsy Res 137:84–94

    Article  CAS  PubMed  Google Scholar 

  18. Bosier B, Muccioli GG, Lambert DM (2013) The FAAH inhibitor URB597 efficiently reduces tyrosine hydroxylase expression through CB1- and FAAH-independent mechanisms. Br J Pharmacol 169:794–807

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Ross RA (2003) Anandamide and vanilloid TRPV1 receptors. Br J Pharmacol 140(5):790–801

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Edwards JG, Gibson HE, Jensen T, Nugent F, Walther C, Blickenstaff J, Kauer JA (2012) A novel non-CB1/TRPV1 endocannabinoid-mediated mechanism depresses excitatory synapses on hippocampal CA1 interneurons. Hippocampus 22:209–221

    Article  CAS  PubMed  Google Scholar 

  21. Aguiar DC, Moreira FA, Terzian AL et al (2014) Modulation of defensive behavior by transient receptor potential vanilloid type-1 (TRPV1) channels. Neurosci Biobehav Rev 46:418–428

    Article  CAS  PubMed  Google Scholar 

  22. Umathe SN, Manna SS, Jain NS (2012) Endocannabinoid analogues exacerbate marble-burying behavior in mice via TRPV1 receptor. Neuropharmacology 62:2024–2033

    Article  CAS  PubMed  Google Scholar 

  23. Ghazizadeh V, Nazıroğlu M (2014) Electromagnetic radiation (Wi-Fi) and epilepsy induce calcium entry and apoptosis through activation of TRPV1 channel in hippocampus and dorsal root ganglion of rats. Metab Brain Dis 29:787–799

    Article  CAS  PubMed  Google Scholar 

  24. Zygmunt PM, Chuang HH, Movahed P, Julius D, Högestätt ED (2000) The anandamide transport inhibitor AM404 activates vanilloid receptors. Eur J Pharmacol 396:39–42

    Article  CAS  PubMed  Google Scholar 

  25. Suemaru K, Yoshikawa M, Aso H, Watanabe M (2018) TRPV1 mediates the anticonvulsant effects of acetaminophen in mice. Epilepsy Res 145:153–159

    Article  CAS  PubMed  Google Scholar 

  26. Alptekin A, Galadari S, Shuba Y, Petroianu G, Oz M (2010) The effects of anandamide transport inhibitor AM404 on voltage-dependent calcium channels. Eur J Pharm 634:10–15

    Article  CAS  Google Scholar 

  27. Caballero FJ, Soler-Torronteras R, Lara-Chica M et al (2015) AM404 inhibits NFAT and NF-κB signaling pathways and impairs migration and invasiveness of neuroblastoma cells. Eur J Pharmacol 746:221–232

    Article  CAS  PubMed  Google Scholar 

  28. Wahab A, Albus K, Heinemann U (2011) Age- and region-specific effects of anticonvulsants and bumetanide on 4-aminopyridine-induced seizure-like events in immature rat hippocampal-entorhinal cortex slices. Epilepsia 52:94–103

    Article  PubMed  Google Scholar 

  29. Fezza F, Marrone MC, Avvisati R et al (2014) Distinct modulation of the endocannabinoid system upon kainic acid-induced in vivo seizures and in vitro epileptiform bursting. Mol Cell Neurosci 62:1–9

    Article  CAS  PubMed  Google Scholar 

  30. Uğuz AC, Nazıroğlu M, Espino J et al (2009) Selenium modulates oxidative stress-induced cell apoptosis in human myeloid HL-60 cells through regulation of calcium release and caspase-3 and – 9 activities. J Membr Biol 232:15–23

    Article  CAS  PubMed  Google Scholar 

  31. Grynkiewicz C, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450

    CAS  Google Scholar 

  32. Espino J, Bejarano I, Redondo PC et al (2010) Melatonin reduces apoptosis induced by calcium signaling in human leukocytes: Evidence for the involvement of mitochondria and Bax activation. J Membr Biol 233:105–118

    Article  CAS  PubMed  Google Scholar 

  33. Espino J, Bejarano I, Paredes SD, Barriga C, Rodríguez AB, Pariente JA (2011) Protective effect of melatonin against human leukocyte apoptosis induced by intracellular calcium overload: relation with its antioxidant actions. J Pineal Res 51:195–206

    Article  CAS  PubMed  Google Scholar 

  34. Cossarizza A, Baccarani-Contri M, Kalashnikova G, Franceschi C (1993) A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1). Biochem Biophys Res Commun 197:40–45

    Article  CAS  PubMed  Google Scholar 

  35. Kelley BG, Thayer SA (2004) Anandamide transport inhibitor AM404 and structurally related compounds inhibit synaptic transmission between rat hippocampal neurons in culture independent of cannabinoid CB1 receptors. Eur J Pharmacol 496:33–39

    Article  CAS  PubMed  Google Scholar 

  36. Yue HY, Fujita T, Kawasaki Y, Kumamoto E (2004) AM404 enhances the spontaneous release of L-glutamate in a manner sensitive to capsazepine in adult rat substantia gelatinosa neurones. Brain Res 1018:283–287

    Article  CAS  PubMed  Google Scholar 

  37. Naderi N, Shafieirad E, Lakpoor D, Rahimi A, Mousavi Z (2005) Interaction between cannabinoid compounds and capsazepine in protection against acute pentylenetetrazole-induced seizure in mice. Iran J Pharm Res 14:115–120

    Google Scholar 

  38. Chemin J, Monteil A, Perez-Reyes E, Nargeot J, Lory P (2001) Direct inhibition of T type calcium channels by the endogenous cannabinoid anandamide. EMBO J 20:7033–7040

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Citraro R, Russo E, Scicchitano F et al (2013) Antiepileptic action of N-palmitoylethanolamine through CB1 and PPAR-γ receptor activation in a genetic model of absence epilepsy. Neuropharmacology 69:115–126

    Article  CAS  PubMed  Google Scholar 

  40. Wallace MJ, Martin BR, DeLorenzo RJ (2002) Evidence for a physiological role of endocannabinoids in the modulation of seizure threshold and severity. Eur J Pharmacol 452:295–301

    Article  CAS  PubMed  Google Scholar 

  41. Sun FJ, Guo W, Zheng DH et al (2013) Increased expression of TRPV1 in the cortex and hippocampus from patients with mesial temporal lobe epilepsy. J Mol Neurosci 49:182–193

    Article  CAS  PubMed  Google Scholar 

  42. Nazıroğlu M (2015) Role of melatonin on calcium signaling and mitochondrial oxidative stress in epilepsy: focus on TRP channels. Tr J Biol 39:813–821

    Google Scholar 

  43. Bahr BA, Karanian DA, Makanji SS, Makriyannis A (2006) Targeting the endocannabinoid system in treating brain disorders. Expert Opin Investig Drugs 15:351–365

    Article  CAS  PubMed  Google Scholar 

  44. Naidoo V, Karanian DA, Vadivel SK et al (2012) Equipotent inhibition of fatty acid amide hydrolase and monoacylglycerol lipase—dual targets of the endocannabinoid system to protect against seizure pathology. Neurotherapeutics 9:801–813

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Soliman E, Van Dross R (2016) Anandamide-induced endoplasmic reticulum stress and apoptosis are mediated by oxidative stress in non-melanoma skin cancer: receptor-independent endocannabinoid signaling. Mol Carcinog 55:1807–1821

    Article  CAS  PubMed  Google Scholar 

  46. Maione S, Bisogno T, de Novellis V et al (2006) Elevation of endocannabinoid levels in the ventrolateral periaqueductal grey through inhibition of fatty acid amide hydrolase affects descending nociceptive pathways via both cannabinoid receptor type 1 and transient receptor potential vanilloid type-1 receptors. J Pharmacol Exp Ther 316(3):969–982

    Article  CAS  PubMed  Google Scholar 

  47. Lee SH, Ledri M, Tóth B et al (2015) Multiple forms of endocannabinoid and endovanilloid signaling regulate the tonic control of GABA release. J Neurosci 35:10039–10057

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Colangeli R, Pierucci M, Benigno A, Campiani G, Butini S, Di Giovanni G (2017) The FAAH inhibitor URB597 suppresses hippocampal maximal dentate afterdischarges and restores seizure-induced impairment of short and long-term synaptic plasticity. Sci Rep 7:11152

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Hakimizadeh E, Oryan S, Hajizadeh Moghaddam A, Shamsizadeh A, Roohbakhsh A (2012) Endocannabinoid system and TRPV1 receptors in the dorsal hippocampus of the rats modulate anxiety-like behaviors. Iran J Basic Med Sci 15:795–802

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Rubino T, Realini N, Castiglioni C et al (2008) Role in anxiety behavior of the endocannabinoid system in the prefrontal cortex. Cereb Cortex 18:1292–1301

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The abstract of the study as poster presentation was submitted to the 6th World Congress of Oxidative Stress, Calcium Signaling and TRP Channels, held 24 and 27 May 2016 in Isparta, Turkey (http://www.cmos.org.tr). The authors wish to thank Dr. Peter Butterworth (Department of Nutrition, King’s College, London, UK) for polishing the English language of the manuscript.

Funding

The study was partially supported by Turkish Scientific and Technological Research Council (TUBITAK-2209-A program).

Author information

Authors and Affiliations

  1. Neuroscience Research Center, Suleyman Demirel University, 32260, Isparta, Turkey

    Mustafa Nazıroğlu

  2. Department of Biophysics, Faculty of Medicine, Suleyman Demirel University, Isparta, Turkey

    Mustafa Nazıroğlu & Bilal Çiğ

  3. Medicine Faculty, Suleyman Demirel University, Isparta, Turkey

    Afife Nur Taner & Esra Balbay

Authors
  1. Mustafa Nazıroğlu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Afife Nur Taner
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Esra Balbay
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bilal Çiğ
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

All authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: MN formulated the hypothesis and was responsible for writing the report. BÇ was responsible for the animal experiments. ANT and EB performed the Ca2+ analyses, apoptosis, and mitochondrial depolarization analyses. MN was responsible from patch-clamp analyses.

Corresponding author

Correspondence to Mustafa Nazıroğlu.

Ethics declarations

Conflict of interest

The authors report no declarations of interest.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Nazıroğlu, M., Taner, A.N., Balbay, E. et al. Inhibitions of anandamide transport and FAAH synthesis decrease apoptosis and oxidative stress through inhibition of TRPV1 channel in an in vitro seizure model. Mol Cell Biochem 453, 143–155 (2019). https://doi.org/10.1007/s11010-018-3439-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11010-018-3439-0

Keywords

  • Anandamide
  • Apoptosis
  • Epilepsy
  • FAAH inhibition
  • TRPV1
  • Oxidative stress