Protective effect of nilvadipine against glutamate neurotoxicity in purified retinal ganglion cells.
We determined the effect of nilvadipine, a dihydropyridine-type calcium channel blocker, in preventing glutamate neurotoxicity in purified retinal ganglion cells (RGCs). RGCs were purified from dissociated rat retinal cells (postnatal days 6-8), using a modified two-step panning method, and cultured in serum-free medium containing neurotrophic factors and forskolin. RGC survival after exposure to glutamate (25 microM) with nilvadipine or other calcium channel blockers was measured by calcein-acetoxymethyl ester staining after 3 days in culture. Changes in the level of intracellular Ca(2+) ([Ca(2+)](i)) were measured with fura-2 fluorescence. Induction of apoptosis was evaluated using the TDT-dUTP terminal nick-end labeling technique. The neurotoxic effects of low doses of glutamate were blocked by a specific alpha-amino-3-dihydro-5-methylisoxazole-4-propionate-kainate receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione (20 microM). Simultaneous application of nilvadipine (1-100 nM) with glutamate protected against glutamate neurotoxicity in a dose-dependent manner. Calcium-imaging experiments showed that the glutamate-evoked [Ca(2+)](i) increase was significantly blocked by nilvadipine (P<0.001), but not nifedipine and diltiazem, in about 50% of RGCs. In addition, the application of nilvadipine significantly reduced glutamate-induced apoptosis (P<0.001). These findings suggest that nilvadipine may partly inhibit glutamate-induced apoptotic cell death by blocking calcium influx via voltage-dependent calcium channels in purified RGCs.
Otori
Yasumasa
Y
Department of Ophthalmology and Visual Science, Osaka University Medical School, Suita, Japan. yotori@ophthal.med.osaka-u.ac.jp
Kusaka
Shunji
S
Kawasaki
Atsushi
A
Morimura
Hiroyuki
H
Miki
Atsuya
A
Tano
Yasuo
Y
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D020746:Calcium Channels, L-Type / Q000378:metabolism; D018929:Cell Culture Techniques; D002470:Cell Survival / Q000187:drug effects; D004110:Diltiazem / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D018698:Glutamic Acid / Q000633:toxicity*; D018696:Neuroprotective Agents / Q000494:pharmacology*; D009543:Nifedipine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D011810:Quinoxalines / Q000494:pharmacology; D051381:Rats; D018091:Receptors, AMPA / Q000378:metabolism*; D018092:Kainic Acid Receptors / Q000378:metabolism*; D012165:Retinal Ganglion Cells / Q000187:drug effects* / Q000378:metabolism
D002118:Calcium; D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; D004110:Diltiazem; D018691:Excitatory Amino Acid Antagonists; D018698:Glutamic Acid; D018696:Neuroprotective Agents; D009543:Nifedipine; D011810:Quinoxalines; D018091:Receptors, AMPA; D018092:Kainic Acid Receptors; C035100:nilvadipine; C056723:FG 9041