Published Online
on December 11, 2003

A more recent version of this article appeared on January 1, 2004

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Submitted on August 28, 2003
Accepted on September 19, 2003

Na⁺/H⁺ Exchanger Inhibitor, SM-20220, Is Protective Against Excitotoxicity in Cultured Cortical Neurons

Yuji Matsumoto MS; Seiji Yamamoto MD, PhD^*; Yasuhiro Suzuki PhD; Takashi Tsuboi PhD; Susumu Terakawa MD, PhD; Naohito Ohashi PhD; and Kazuo Umemura MD, PhD

From the Department of Pharmacology (Y.M., Y.S., K.U.) and Photon Medical Research Center (Y.M., S.Y., T.T., S.T.), Hamamatsu University School of Medicine, Hamamatsu, and Research Division (N.O.), Sumitomo Pharmaceuticals Co Ltd, Osaka, Japan.

^* To whom correspondence should be addressed. E-mail: seijiy{at}hama-med.ac.jp.

Background and Purpose--Recently, it has been reported that Na⁺/H⁺ exchanger (NHE) inhibitors demonstrated protective effects on ischemia/reperfusion brain injury in animal models. However, the mechanisms by which the neurons were protected against ischemic insult remain unclear. To reveal the cellular mechanism of the NHE inhibitor on the neuronal death, we examined the effects of a selective NHE inhibitor, SM-20220 (N-[aminoiminomethyl]-1-methyl-1H-indole-2-carboxamide methanesulfonate), on glutamate-induced neuronal death in rat cortical culture.

Methods--Cortical neurons were prepared from 1-day old rats, and cultured on the glass-based dishes. Glutamate-induced neuronal death was assessed by staining the cells with propidium iodide. Morphological changes in the neurons were observed with a video-enhanced contrast-differential interference contrast microscope. The intracellular calcium concentration ([Ca²⁺]_i) and the intracellular pH (pH_i) were measured by fluorescence imaging with a confocal laser microscope using fluo-3/acetoxymethylester (AM) and 2', 7'-bis-2-carboxy-ethyl-5(6)-carboxyfluorescein (BCECF)/AM as a fluorescent dye, respectively.

Results--SM-20220 (0.3 to 30 nmol/L) dose-dependently attenuated glutamate (300 µmol/L)-induced neuronal death over a period of 6 hours, and inhibited the acute cellular swelling following glutamate (500 µmol/L) exposure. Dual peaks of [Ca²⁺]_i rise were observed at 5 and 12 minutes after glutamate (500 µmol/L) exposure, followed by a persistent rise. SM-20220 suppressed the persistent [Ca²⁺]_i increase. SM-20220 inhibited intracellular acidification following glutamate (500 µmol/L) exposure. All of the events induced by glutamate were also inhibited by the N-methyl-D-aspartate receptor antagonist, MK-801, indicating the death process was excitotoxicity.

Conclusions--NHE inhibitor is neuroprotective through inhibition of both persistent [Ca²⁺]_i increase and acidification in excitotoxicity.

Key words: excitotoxicity • glutamates • Na⁺/H⁺ exchanger • neuronal death • rats

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