Na+/H+ exchanger inhibitor, SM-20220, is protective against excitotoxicity in cultured cortical neurons.
OBJECTIVE
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 (pHi) 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 micromol/L)-induced neuronal death over a period of 6 hours, and inhibited the acute cellular swelling following glutamate (500 micromol/L) exposure. Dual peaks of [Ca2+]i rise were observed at 5 and 12 minutes after glutamate (500 micromol/L) exposure, followed by a persistent rise. SM-20220 suppressed the persistent [Ca2+]i increase. SM-20220 inhibited intracellular acidification following glutamate (500 micromol/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.
Matsumoto
Yuji
Y
Department of Pharmacology, Hamamatsu University School of Medicine, Hamamatsu, Japan.
Yamamoto
Seiji
S
Suzuki
Yasuhiro
Y
Tsuboi
Takashi
T
Terakawa
Susumu
S
Ohashi
Naohito
N
Umemura
Kazuo
K
D000577:Amides / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D004305:Dose-Response Relationship, Drug; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D018698:Glutamic Acid / Q000633:toxicity; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007211:Indoles / Q000494:pharmacology*; D007424:Intracellular Fluid / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D018696:Neuroprotective Agents / Q000494:pharmacology*; D009498:Neurotoxins / Q000633:toxicity; D051381:Rats; D017208:Rats, Wistar; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors*
D000577:Amides; D018691:Excitatory Amino Acid Antagonists; D007211:Indoles; D018696:Neuroprotective Agents; D009498:Neurotoxins; C401341:SM 20220; D017923:Sodium-Hydrogen Exchangers; D018698:Glutamic Acid; D002118:Calcium