Elsevier

Life Sciences

Volume 78, Issue 3, 5 December 2005, Pages 294-300
Life Sciences

Blockade of 5-HT3 receptor with MDL7222 and Y25130 reduces hydrogen peroxide-induced neurotoxicity in cultured rat cortical cells

https://doi.org/10.1016/j.lfs.2005.04.043Get rights and content

Abstract

The present study was performed to examine the neuroprotective effects of 5-hydroxytryptamine (5-HT)3 receptor antagonists against hydrogen peroxide (H2O2)-induced neurotoxicity using cultured rat cortical neurons. Pretreatment of 5-HT3 receptor antagonists, tropanyl-3,5-dichlorobenzoate (MDL72222, 0.1 and 1 μM) and N-(1-azabicyclo[2.2.2.]oct-3-yl)-6-chloro-4-ethyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-8-carboxamide hydrochloride (Y25130, 0.5 and 5 μM), significantly inhibited the H2O2 (100 μM)-induced neuronal cell death as assessed by a MTT assay and the number of apoptotic nuclei, evidenced by Hoechst 33342 staining. The protective effects of MDL72222 (1 μM) and Y25130 (5 μM) were completely blocked by the simultaneous treatment with 100 μM 1-phenylbiguanide, a 5-HT3 receptor agonist, indicating that the protective effects of these compounds were due to 5-HT3 receptor blockade. In addition, MDL72222 (1 μM) and Y25130 (5 μM) inhibited the H2O2 (100 μM)-induced elevation of cytosolic Ca2+ concentration ([Ca2+]c) and glutamate release, generation of reactive oxygen species (ROS), and caspase-3 activity. These results suggest that the activation of the 5-HT3 receptor may be partially involved in H2O2-induced neurotoxicity, by membrane depolarization for Ca2+ influx. Therefore, the blockade of 5-HT3 receptor with MDL72222 and Y25130 may ameliorate the H2O2-induced neurotoxicity by interfering with the increase of [Ca2+]c, and then by inhibiting glutamate release, generation of ROS and caspase-3 activity.

Introduction

Formation of reactive oxygen species (ROS) has been proposed to be an important step leading to neuronal death related to a variety of cerebral diseases, e.g. stroke, amyotropic lateral sclerosis, Alzheimer's disease and Parkinson's disease (Olanow, 1993). Therefore, in vitro hydrogen peroxide (H2O2) toxicity has become a well-established model for the oxidative stress on neurons. Many researches demonstrated the involvement of glutamate in H2O2-induced neurotoxicity in cultured neurons (Gardner et al., 1996). H2O2 and O2 inhibited the uptake of glutamate and enhanced the release of glutamate, resulting in NMDA receptor overstimulation (Volterra et al., 1994, Mailly et al., 1999). The neurotoxic effects of H2O2 were strongly reduced by antagonists of NMDA receptors and enhanced in the absence of Mg2+ (Mailly et al., 1999). There are some reports on H2O2-induced intracellular Ca2+ concentration ([Ca2+]i) increase (Whittemore et al., 1995). NMDA receptor is a ligand-gated/voltage-sensitive cation channel, especially highly permeable to Ca2+. Calcium influx through NMDA receptor-coupled Ca2+ channel appears to be a critical role in the H2O2-induced neurotoxicity (Mailly et al., 1999). Moreover, Ca2+ signals activate enzymes which lead to further ROS generation (e.g. xanthine oxidase, nitric oxide synthase, phospholipase A2); conversely, ROS generation can facilitate [Ca2+]i increases by damaging the [Ca2+]i regulatory mechanism and activating Ca2+ release from intracellular Ca2+ stores (Duffy and MacViar, 1996). However, the clinical benefit of NMDA receptor antagonists and direct blockers of neuronal Ca2+ channels is debatable, since they lack convincing effectiveness or have serious side-effects (Ferger and Krieglstein, 1996, Li et al., 2002).

5-HT3 receptor antagonists were shown to enhance cholinergic function and this corroborated behavioral evidence that they had cognition-enhancing properties in experimental animals (Costal and Naylor, 1994). Moreover, ondansetron, a 5-HT3 receptor antagonist, was reported to attenuate the deficit in various learned behaviors caused by systemically administered scopolamine (Carli et al., 1997). It was also demonstrated that the blockade of 5-HT3 receptors plays a neuroprotective role in ischemia-induced damage (Kagami-ishi et al., 1992). Given the potential pharmacological activity of 5-HT3 receptor antagonists, it seems reasonable to explore the neuroprotective effects of 5-HT3 receptor antagonists against H2O2-induced apoptotic death and to study associated potential underlying mechanisms. Therefore, our study aims at determining whether blockade of 5-HT3 receptor with tropanyl-3,5-dichlorobenzoate (MDL72222), N-(1-azabicyclo[2.2.2.]oct-3-yl)-6-chloro-4-ethyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-8-carboxamide hydrochloride (Y25130) is able to protect the neuronal cells against H2O2-induced neurotoxicity in cultured rat cortical cells.

Section snippets

Materials

Tropanyl-3,5-dichlorobenzoate (MDL72222) and N-(1-azabicyclo[2.2.2.]oct-3-yl)-6-chloro-4-ethyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-8-carboxamide hydrochloride (Y25130) and 1-phenylbiguanide hydrochloride (PGB) were purchased from Tocris Cookson Inc. (St. Ballwin, Mo, USA). (5R,10S)-(+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine (MK-801) was purchased from RBI (Natick, MA, USA). H2O2, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT), o-phthaldialdehyde

5-HT3 receptor antagonists protect neurons against cell death induced by H2O2

To assess H2O2-induced neuronal cell death, the MTT assay was performed. In previous report, we demonstrated that H2O2 over the concentration range of 10–200 μM produced a concentration-dependent reduction of cell viability in cultured cortical neurons (Lee et al., 2005). For the present experiments, 100 μM of H2O2 was used. MTT reduction rate decreased to 59.4 ± 1.4% with the treatment with 100 μM H2O2. MDL72222 (0.01, 0.1 and 1 μM) and Y25130 (0.05, 0.5 and 5 μM) concentration-dependently

5-HT3 receptor antagonists inhibit H2O2-induced caspase-3 protein activation

Caspase-3, the 32-kDa protease constitutively expressed by many cell types and tissues, is implicated in apoptosis promoted by different death stimuli. Caspase-3 protein expression was evaluated by Western blotting. In 100 μM H2O2-treated cells, caspase-3 activity markedly increased compared to control cultures. MDL72222 (1 μM) and Y25130 (5 μM) significantly blocked the H2O2-induced increase of caspase-3 immunoreactivity. However, the blockade of 100 μM H2O2-induced caspase-3 expression by the

Discussion

The present study provides evidence that oxidative stress-induced injury to rat cortical neurons can be prevented by 5-HT3 receptor antagonists. 5-HT3 receptor antagonists, MDL72222 and Y25130, were able to reduce the H2O2-induced neuronal apoptotic death, [Ca2+]c increase, glutamate release, ROS generation and caspase-3 activation. The nearly complete reversal of the anti-apoptotic effects and inhibitory effects on caspase-3 activation of the 5-HT3 receptor antagonists by the receptor agonist,

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