Metzger F, Kulik A, Sendtner M, Ballanyi K
Klinische Forschergruppe Neuroregeneration, Neurologische Klinik, Universität Würzburg, D-97080 Wurzburg, Germany.
J Neurophysiol. 2000 Jan;83(1):50-9. doi: 10.1152/jn.2000.83.1.50.
Intracellular Ca(2+) (Ca(2+)) was fluorometrically measured with fura-2 in lumbar motoneurons of acutely isolated spinal cord slices from embryonic rats. In ester-loaded cells, bath-applied glutamate (3 microM to 1 mM) evoked a Ca(2+) increase by up to 250 nM that was abolished by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) plus 2-amino-5-phosphonovalerate (APV). CNQX or APV alone reduced the response by 82 and 25%, respectively. The glutamatergic agonists kainate (KA), quisqualate (QUI), and S-alpha-amino-3-hydroxy-5-methyl-4-isoxalone (S-AMPA) evoked a similar Ca(2+) transient as glutamate. N-methyl-D-aspartate (NMDA) was only effective to increase Ca(2+) in Mg(2+)-free saline, whereas [1S,3R]-1-aminocyclopentane-1,3-dicarboxylic acid ([1S,3R]-ACPD) had no effect. The glutamate-induced Ca(2+) rise was suppressed in Ca(2+)-free superfusate. Depletion of Ca(2+) stores with cyclopiazonic acid (CPA) did not affect the response. Thirty-six percent of the Ca(2+) increase in response to membrane depolarization induced by a 50 mM K(+) solution persisted on combined application of the voltage-gated Ca(2+) channel blockers nifedipine, omega-conotoxin-GVIA and omega-agatoxin-IVA. In fura-2 dialyzed motoneurons, the glutamate-induced Ca(2+) increase was attenuated by approximately 70% after changing from current to voltage clamp. Forty percent of the remaining Ca(2+) transient and 20% of the concomitant inward current of 0.3 nA were blocked by Joro spider toxin-3 (JSTX). The results show that voltage-gated Ca(2+) channels, including a major portion of R-type channels, constitute the predominant component of glutamate-induced Ca(2+) rises. NMDA and Ca(2+)-permeable KA/AMPA receptors contribute about equally to the remaining component of the Ca(2+) rise. The results substantiate previous assumptions that Ca(2+) influx through JSTX-sensitive KA/AMPA receptors is involved in (trophic) signaling in developing motoneurons.
采用荧光法，运用fura - 2对急性分离的胚胎大鼠脊髓切片腰段运动神经元内的细胞内钙离子浓度（[Ca(2+)]i）进行测量。在酯负载细胞中，浴槽施加谷氨酸（3 microM至1 mM）可使[Ca(2+)]i升高多达250 nM，而6 - 氰基 - 7 - 硝基喹喔啉 - 2,3 - 二酮（CNQX）加2 - 氨基 - 5 - 膦酰基戊酸（APV）可消除该升高。单独使用CNQX或APV分别使反应降低82%和25%。谷氨酸能激动剂海人酸（KA）、quisqualate（QUI）和S - α - 氨基 - 3 - 羟基 - 5 - 甲基 - 4 - 异恶唑酮（S - AMPA）诱发的[Ca(2+)]i瞬变与谷氨酸相似。N - 甲基 - D - 天冬氨酸（NMDA）仅在无镁盐溶液中有效增加[Ca(2+)]i，而[1S,3R] - 1 - 氨基环戊烷 - 1,3 - 二羧酸（[1S,3R] - ACPD）无作用。谷氨酸诱导的[Ca(2+)]i升高在无钙灌流液中受到抑制。用环匹阿尼酸（CPA）耗尽钙库不影响反应。在联合应用电压门控性Ca(2+)通道阻滞剂硝苯地平、ω - 芋螺毒素 - GVIA和ω - 阿加毒素 - IVA时，对50 mM K(+)溶液诱导的膜去极化反应中，[Ca(2+)]i升高的36%仍然存在。在fura - 2透析的运动神经元中，从电流钳转换为电压钳后，谷氨酸诱导的[Ca(2+)]i升高减弱约70%。剩余[Ca(2+)]i瞬变的40%以及伴随的0.3 nA内向电流的20%被Joro蜘蛛毒素 - 3（JSTX）阻断。结果表明，电压门控性Ca(2+)通道，包括大部分R型通道，构成了谷氨酸诱导的[Ca(2+)]i升高的主要成分。NMDA和钙通透型KA/AMPA受体对Ca(2+)升高的剩余成分贡献大致相等。这些结果证实了先前的假设，即通过JSTX敏感的KA/AMPA受体的Ca(2+)内流参与发育中运动神经元的（营养性）信号传导。
