Abstract
Pyridoxine (vitamin B6) protects neurons against neurotoxicity. An excessive release of glutamate is widely considered to be one of the molecular mechanisms of neuronal damage in several neurological diseases. We investigated whether pyridoxine affected glutamate release in rat cerebral cortex nerve terminals (synaptosomes). Pyridoxine inhibited the release of glutamate that was evoked by exposing synaptosomes to the K+ channel blocker 4-aminopyridine (4-AP), and this phenomenon was concentration-dependent. Inhibition of glutamate release by pyridoxine was prevented by the vesicular transporter inhibitor bafilomycin A1, or by chelating intraterminal Ca2+, but was insensitive to DL-threo-β-benzyl-oxyaspartate, a glutamate transporter inhibitor. Pyridoxine did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization. Examination of the effect of pyridoxine on cytosolic [Ca2+] revealed that diminution of glutamate release could be attributed to a reduction in voltage-dependent Ca2+ influx. Consistent with this, the pyridoxine-mediated inhibition of glutamate release was completely prevented by blocking the N- and P/Q-type Ca2+ channels, but not by blocking intracellular Ca2+ release or Na+/Ca2+ exchange. Furthermore, the pyridoxine effect on 4-AP-evoked glutamate release was abolished by the protein kinase C (PKC) inhibitors bisindolylmaleimide I (GF109203X) or bisindolylmaleimide IX (Ro318220), and pyridoxine significantly decreased the 4-AP-induced phosphorylation of PKC, PKCα, and myristoylated alanine-rich C kinase substrate. Together, these results suggest that pyridoxine inhibits glutamate release from rat cortical synaptosomes, through the suppression of presynaptic voltage-dependent Ca2+ entry and PKC activity.
- VDCC, voltage-dependent Ca2+ channel
- 4-AP, 4-aminopyridine
- [Ca2+]C, cytosolic free Ca2+ concentration
- DiSC3(5), 3',3',3'-dipropylthiadicarbocyanine iodide
- DL-TBOA, DL-threo-β-benzyl-oxy aspartate
- Fura-2-AM, fura-2-acetoxymethyl ester
- GDH, glutamate dehydrogenase
- HBM, HEPES buffer medium
- BSA, bovine serum albumin
- BAPTA-AM, 1,2-bis (2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester
- ω-CgTX GVIA, ω-conotoxin GVIA
- ω-AgTX IVA, ω-agatoxin IVA
- ω-CgTX MVIIC, ω-conotoxin MVIIC
- MARCKS, myristoylated alanine-rich C kinase substrate
- PKC, protein kinase C
- GF109203X, bisindolylmaleimide I
- Ro318220, bisindolylmaleimide IX
- CGP37157, 7-chloro-5-(2-chloropheny)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one
- PD98059, 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one)
- PLP, pyridoxal phosphate
- ANOVA, analysis of variance
- MAP, mitogen-activated protein.
Footnotes
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This work was supported by the National Science Council of Taiwan, Republic of China [Grant NSC 96-2628-B-020-001 MY3]; and the Cardinal Tien Hospital [Grant CTH-98-1-2A30].
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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ABBREVIATIONS:
- Received April 21, 2009.
- Accepted July 22, 2009.
- © 2009 by The American Society for Pharmacology and Experimental Therapeutics
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