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

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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.

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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

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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).

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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

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  1. Mustafa Nazıroğlu
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  2. Afife Nur Taner
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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.

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Correspondence to Mustafa Nazıroğlu.

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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

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