Blockade of 5-HT(3) receptor with MDL 72222 and Y 25130 reduces beta-amyloid protein (25--35)-induced neurotoxicity in cultured rat cortical neurons

The present study was performed to examine neuroprotective effects of 5-hydroxytryptamine (5-HT)₃ receptor antagonists against β-amyloid protein (25–35)-, a synthetic 25–35 amyloid peptide, induced neurotoxicity using cultured rat cortical neurons. β-Amyloid protein (25–35) produced a concentration-dependent reduction of cell viability, which was significantly reduced by (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzoa,d] cyclohepten-5,10-imine (MK-801), an N-methyl-d-aspartate (NMDA) receptor antagonist, verapamil, an L-type Ca²⁺ channel blocker, and N^G-nitro-l-arginine methyl ester (l-NAME), a nitric oxide synthase inhibitor. The 5-HT₃ receptor antagonists, tropanyl-3,5-dichlorobenzoate (MDL72222, 0.1–10 μM) and N-(1-azabicyclo2.2.2.]oct-3-yl)-6-chloro-4-ethyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-8-carboxamide hydrochloride (Y25130, 0.05–5 μM), decreased the β-amyloid protein (25–35) (10 μM)-induced neuronal cell death as assessed by a colorimetric 3-4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and the number of apoptotic nuclei, evidenced by Hoechst 33342 staining. MDL72222 and Y25130 inhibited the β-amyloid protein (25–35) (10 μM)-induced elevation of cytosolic Ca²⁺ concentration (Ca²⁺]_c) and glutamate release, generation of reactive oxygen species, and caspase-3 activity. These neuroprotective effects of MDL72222 (10 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. These results suggest that the activation of the 5-HT₃ receptor may be partially involved in β-amyloid protein-induced neurotoxicity, by membrane depolarization for Ca²⁺ influx. Therefore, the blockade of 5-HT₃ receptor with MDL72222 and Y25130, may ameliorate the β-amyloid protein-induced neurotoxicity by interfering with the increase of Ca²⁺]_c, and then by inhibiting glutamate release, generation of reactive oxygen species and caspase-3 activity.