Marino Silvia, Marani Luca, Nazzaro Cristiano, Beani Lorenzo, Siniscalchi Anna
Department of Clinical and Experimental Medicine, Section of Pharmacology, University of Ferrara, Via Fossato di Mortara 17-19, 44100 Ferrara, Italy.
Neurotoxicology. 2007 May;28(3):622-9. doi: 10.1016/j.neuro.2007.01.005. Epub 2007 Jan 20.
An intracellular calcium (Ca(2+)) increase is involved in sodium azide (NaN(3))-induced neurotoxicity, an in vitro model of brain ischemia. In this study the questions of possible additional sources of calcium influx, besides glutamate receptor activation, and of the time-course of NaN(3) effects have been addressed by measuring Ca(2+) in rat primary cortical cultures with the FURA-2 method. Basal Ca(2+) of neuronal populations was concentration-dependently increased 30 min, but not 24h, after a 10-min NaN(3) (3-30 mM) treatment; conversely, the net increase induced by electrical stimulation (10Hz, 10s) was consistently reduced. All the above effects depended on glutamate release and consequent NMDA receptor activation, since the NMDA antagonist MK-801 (1 microM) prevented them, and the spontaneous efflux of [(3)H]-d-aspartate from superfused neurons was concentration-dependently increased by NaN(3). In single neuronal cells, NaN(3) application progressively and concentration-dependently increased Ca(2+) (to 177+/-5% and 249+/-7% of the controls, 4 and 12 min after a 10mM-treatment, respectively). EGTA (5mM) pretreatment reduced the effect of 10mM NaN(3) (to 118+/-5% at 4 min, and to 148+/-10% at 12 min, respectively), while 1 microM cyclosporin A did not. Both MK-801 and CNQX (a non-NMDA glutamate antagonist, 10 microM) prevented NaN(3) effect at 4 min (to 147+/-8% and 153+/-5%, respectively), but not at 12 min after NaN(3) treatment. Conversely, 10 microM verapamil and 0.1 microM omega-conotoxin (L- and N-type calcium channel blockers, respectively) significantly attenuated NaN(3) effects at 12 min (to 198+/-8% and 164+/-5%, respectively), but not at 4 min; the P/Q-type calcium channel blocker, agatoxin, 0.3 microM, was ineffective. These findings show that the predominant source of calcium increase induced by NaN(3) is extracellular, involving glutamate receptor activation in a first step and calcium channel (mainly of the N-type) opening in a second step.
细胞内钙（Ca(2+)）升高参与了叠氮化钠（NaN(3)）诱导的神经毒性，这是一种脑缺血的体外模型。在本研究中，通过用FURA-2方法测量大鼠原代皮质培养物中的Ca(2+)，探讨了除谷氨酸受体激活外可能的额外钙内流来源问题以及NaN(3)作用的时间进程。在10分钟的NaN(3)（3 - 30 mM）处理后30分钟，神经元群体的基础Ca(2+)呈浓度依赖性升高，但24小时后未升高；相反，电刺激（10Hz，10秒）诱导的净升高持续降低。上述所有效应均依赖于谷氨酸释放及随后的NMDA受体激活，因为NMDA拮抗剂MK-801（1 microM）可阻止这些效应，并且NaN(3)可使灌流神经元中[(3)H]-d-天冬氨酸的自发外流呈浓度依赖性增加。在单个神经元细胞中，施加NaN(3)可使Ca(2+)逐渐且浓度依赖性升高（在10 mM处理后4分钟和12分钟分别达到对照的177±5%和249±7%）。EGTA（5 mM）预处理可降低10 mM NaN(3)的作用（4分钟时降至118±5%；12分钟时降至148±10%），而1 microM环孢素A则无效。MK-801和CNQX（一种非NMDA谷氨酸拮抗剂，10 microM）均可在4分钟时阻止NaN(3)的作用（分别降至对照的147±8%和153±5%），但在NaN(3)处理后第12分钟时则不能。相反，10 microM维拉帕米和0.1 microM ω-芋螺毒素（分别为L型和N型钙通道阻滞剂）在12分钟时可显著减弱NaN(3)的作用（分别降至198±8%和164±5%）但在4分钟时则不能；0.3 microM的P/Q型钙通道阻滞剂阿加毒素无效。这些发现表明，NaN(3)诱导的钙升高的主要来源是细胞外，第一步涉及谷氨酸受体激活，第二步涉及钙通道（主要是N型）开放。
