Nitrobenzylthioinosine mimics adenosine to attenuate the epileptiform discharge of hippocampal neurons from epileptic rats

Abstract

Nitrobenzylthioinosine (NBTI), a specific inhibitor of type 1 equilibrative nucleoside transporter, could regulate the extracellular adenosine concentration and have protective roles in seizures. However, the protection mechanism of NBTI in seizures remains poorly understood. Here, the expression pattern and subcellular distribution of adenosine A1 receptor were detected by Western blot analysis and double-labeling immunofluorescence staining in Lithium Chloride-Pilocarpine induced epileptic rat model. At 24 h after pilocarpine induced rat seizures, hippocampal slices were prepared and the evoked excitatory postsynaptic currents (eEPSCs) amplitude of pyramidal neurons in hippocampus CA1 region was recorded using whole-cell patch clamp. In vivo, compared to control group, Western blotting analysis showed that the expression of adenosine A1 receptor protein was increased at 24 h and 72 h after seizure, didn't change at 0 min and 1 w, and decreased at 2 w. Double-label immunofluorescence revealed that adenosine A1 receptor was mainly expressed in the membrane and cytoplasm of neurons. In Vitro, adenosine decreased the eEPSCs amplitude of pyramidal neurons in hippocampus CA1 region, NBTI also had the same effect. Meantime, NBTI could further inhibit eEPSCs amplitude on the basis of lower concentration adenosine (50μM), and adenosine A1 receptor inhibitor DPCPX partially reversed this effect. Taken together, we confirmed that the expression of adenosine A1 receptor protein was increased in the early seizures and decreased in the late seizures. At the same time, NBTI mimics adenosine to attenuate the epileptiform discharge through adenosine A1 receptor, which might provide a novel therapeutic approach toward the control of epilepsy.

Inhibition of p38 MAPK regulates epileptic severity by decreasing expression levels of A1R and ENT1

“…and hippocampal injections of nBTi, a selective enT1 inhibitor, decreased the number of seizures and prolonged the latency of the first seizure in rats, indicating that enT1 plays an important role in the development of epilepsy (29). Therefore, enT1 may be a novel therapeutic target for the control of epileptic seizures in the clinic (22,28). Based on the results of western blot analysis in the present study, enT1 expression levels 3 days after seizure induction were significantly lower in the SB203580 group than in the epilepsy group, showing that inhibition of the p38 MaPK signaling pathway modified ENT1 expression levels in epileptic rats.…”

“…Our previous study (20) confirmed that the specific p38 MaPK inhibitor SB203580 alleviated epileptic seizures in Se rats. nitrobenzothioinosine (nBTi), a selective inhibitor of enT1 (21), blocks the transport of adenosine into cells and increases extracellular adenosine levels, thus exhibiting a protective effect against epileptic seizures (22). a number of studies have revealed that a1r, a member of the adenosine system, has significant anticonvulsant effects (13,23).…”

Metabolic Aspects of Adenosine Functions in the Brain

“…In several epilepsy models, adenosine has been shown to exert an anticonvulsant effect (Boison, 2012;Masino et al, 2014), which appears to be mainly mediated by A1R (Li et al, 2007). ENT1 may be considered a therapeutic target for the control of epileptic seizures (Huang et al, 2017). Indeed, intraperitoneal and hippocampal injections of nitrobenzythioinosine (NBTI), a specific inhibitor of ENT1, decreased the number of seizures in rats (Xu et al, 2015), while blood brain barrier permeable ENT1 inhibitors have shown anti-epileptic effects in different mouse models (Ho et al, 2020).…”

Mechanism of Robo1 in the pentylenetetrazol‐kindled epilepsy mouse model

“…Immunofluorescence was performed as previously described by our laboratory 19 . Briefly, brain tissue sections were fixed in 4% polyformaldehyde for 1 min.…”