Numakawa T, Matsumoto T, Adachi N, Yokomaku D, Kojima M, Takei N, Hatanaka H
Division of Protein Biosynthesis, Institute for Protein Research, Osaka University, Suita, Osaka, Japan.
J Neurosci Res. 2001 Oct 1;66(1):96-108. doi: 10.1002/jnr.1201.
We reported previously that BDNF induced glutamate release was dependent on intracellular Ca(2+) but not extracellular Ca(2+) in cerebellar neurons (Numakawa et al., 1999). It was revealed that the release was through a non-exocytotic pathway (Takei et al., 1998; Numakawa et al., 1999). In the present study, we monitored the dynamics of intracellular Ca(2+) and Na(+) in cerebellar neurons, and investigated the possibility of reverse transport of glutamate mediated by BDNF. As reported, BDNF increased the intracellular Ca(2+) level. We found that the Ca(2+) increase induced by BDNF was completely blocked by xestospongin C, an IP(3) receptor antagonist, and U-73122, a PLC-gamma inhibitor. Xestospongin C and U-73122 also blocked the BDNF-dependent glutamate release, suggesting that the BDNF-induced transient increase of Ca(2+) through the activation of the PLC-gamma/ IP(3) pathway was essential for the glutamate release. We found that BDNF induced a Na(+) influx. This was blocked by treatment with TTX. U-73122 and xestospongin C blocked the BDNF-induced Na(+) influx, suggesting that the Na(+)influx required the BDNF-induced Ca(2+) increase. Next, we examined the possibility that a co-transporter of Na(+) and glutamate was involved in the BDNF-induced glutamate release. BDNF-induced glutamate release was blocked by L-trans-pyrollidine-2,4-dicalboxylic acid (t-PDC), a glutamate transporter inhibitor, whereas neither the 4-aminopyridine (4AP)- nor high potassium (HK(+))-induced release was blocked by t-PDC. In addition, DL-threo-beta-benzyloxyaspartate (DL-TBOA) also blocked the BDNF-mediated glutamate release, suggesting that reverse transport of glutamate may be involved. All the results therefore suggest that Na(+)-dependent reverse transport contributes to BDNF-mediated transmitter release through the PLC-gamma/IP(3)-mediated Ca(2+) signaling.
我们之前报道过，在小脑神经元中，脑源性神经营养因子（BDNF）诱导的谷氨酸释放依赖于细胞内钙离子（Ca(2+)），而非细胞外钙离子（Ca(2+)）（沼川等人，1999年）。研究表明，这种释放是通过非胞吐途径进行的（武井等人，1998年；沼川等人，1999年）。在本研究中，我们监测了小脑神经元内钙离子（Ca(2+)）和钠离子（Na(+)）的动态变化，并研究了BDNF介导的谷氨酸逆向转运的可能性。如前所述，BDNF提高了细胞内钙离子（Ca(2+)）水平。我们发现，BDNF诱导的钙离子（Ca(2+)）增加被肌醇三磷酸（IP(3)）受体拮抗剂西司他汀C和磷脂酶C-γ（PLC-γ）抑制剂U-73122完全阻断。西司他汀C和U-73122也阻断了BDNF依赖的谷氨酸释放，这表明BDNF通过激活PLC-γ/IP(3)途径诱导的钙离子（Ca(2+)）瞬时增加对于谷氨酸释放至关重要。我们发现BDNF诱导了钠离子（Na(+)）内流。这被河豚毒素（TTX）处理所阻断。U-73122和西司他汀C阻断了BDNF诱导的钠离子（Na(+)）内流，这表明钠离子（Na(+)）内流需要BDNF诱导的钙离子（Ca(2+)）增加。接下来，我们研究了钠离子（Na(+)）和谷氨酸共转运体参与BDNF诱导的谷氨酸释放的可能性。BDNF诱导的谷氨酸释放被谷氨酸转运体抑制剂L-反式-吡咯烷-2,4-二羧酸（t-PDC）阻断，而4-氨基吡啶（4AP）或高钾（HK(+)）诱导的释放均未被t-PDC阻断。此外，DL-苏式-β-苄氧基天冬氨酸（DL-TBOA）也阻断了BDNF介导的谷氨酸释放，这表明可能涉及谷氨酸的逆向转运。因此，所有结果表明，钠离子（Na(+)）依赖的逆向转运通过PLC-γ/IP(3)介导的钙离子（Ca(2+)）信号传导促进了BDNF介导的递质释放。
