Parks T N, Artman L D, Alasti N, Nemeth E F
Natural Product Sciences, Inc., Salt Lake City, UT 84108.
Brain Res. 1991 Jun 21;552(1):13-22. doi: 10.1016/0006-8993(91)90653-d.
The concentration of intracellular free Ca2+ ([Ca2+]i) was measured in rat cerebellar granule cells using the fluorescent indicator fura-2. Culturing the cells as monolayers on plastic squares which could be placed into cuvettes allowed measurements of [Ca2+]i to be performed on large and homogeneous populations of CNS neurons. Granule cells so cultured maintained low levels of [Ca2+]i (around 90 nM) which increased promptly upon the addition of various excitatory amino acids including N-methyl-D-aspartate (NMDA). Increases in [Ca2+]i elicited by NMDA were inhibited by Mg2+ (1 mM) and often potentiated by glycine (1 microM). The addition of TTX or strychnine (5 microM each) did not alter responses to NMDA or NMDA plus glycine. Cytosolic Ca2+ responses to NMDA/glycine were dependent on the presence of extracellular Ca2+ and were unaffected by concentrations of nifedipine or verapamil that blocked increases in [Ca2+]i elicited by K+ depolarization. Responses elicited by NMDA/glycine were inhibited competitively by 2-amino-5-phosphonovalerate or 3-((+-)-2-carboxypiperazin-4-yl)-propyl-1- phosphonic acid and non-competitively by MK-801 or Mg2+. HA-966 and 7-chlorokynurenate inhibited responses to NMDA alone and blocked competitively the potentiating effects of glycine. The results demonstrate NMDA-mediated increases in [Ca2+]i in cerebellar granule cells that arise solely from influx of extracellular Ca2+ through dihydropyridine-insensitive channels. The strict dependence of the NMDA-evoked response on extracellular Ca2+ provides little evidence for a coupling of NMDA receptors to inositol phosphate metabolism and mobilization of intracellular Ca2+. The effect of various agents on NMDA/glycine-induced increases in [Ca2+]i parallels their effects on ligand binding to or current flow through the NMDA receptor-channel complex. The measurement of cytosolic Ca2+ in this preparation of neuronal cells thus appears especially well suited for assessing, on a functional level, the regulation of NMDA receptors in the CNS.
利用荧光指示剂fura-2测定大鼠小脑颗粒细胞内游离Ca2+（[Ca2+]i）的浓度。将细胞单层培养在可放入比色皿的塑料方块上，这样就能对大量且均匀的中枢神经系统神经元群体进行[Ca2+]i的测量。如此培养的颗粒细胞维持着较低水平的[Ca2+]i（约90 nM），在加入包括N-甲基-D-天冬氨酸（NMDA）在内的各种兴奋性氨基酸后，[Ca2+]i会迅速升高。NMDA引起的[Ca2+]i升高受到Mg2+（1 mM）的抑制，且常被甘氨酸（1 microM）增强。加入河豚毒素或士的宁（各5 microM）不会改变对NMDA或NMDA加甘氨酸的反应。胞质Ca2+对NMDA/甘氨酸的反应依赖于细胞外Ca2+的存在，并且不受硝苯地平或维拉帕米浓度的影响，这些药物会阻断K+去极化引起的[Ca2+]i升高。NMDA/甘氨酸引起的反应被2-氨基-5-磷酸戊酸或3-((+-)-2-羧基哌嗪-4-基)-丙基-1-膦酸竞争性抑制，被MK-801或Mg2+非竞争性抑制。HA-966和7-氯犬尿氨酸单独抑制对NMDA的反应，并竞争性阻断甘氨酸的增强作用。结果表明，NMDA介导的小脑颗粒细胞内[Ca2+]i升高仅源于细胞外Ca2+通过对二氢吡啶不敏感的通道内流。NMDA诱发反应对细胞外Ca2+的严格依赖性几乎没有提供证据表明NMDA受体与肌醇磷酸代谢及细胞内Ca2+动员存在偶联。各种药物对NMDA/甘氨酸诱导的[Ca2+]i升高的影响与其对配体与NMDA受体通道复合物结合或电流通过的影响相似。因此，在这种神经元细胞制备物中测量胞质Ca2+似乎特别适合在功能水平上评估中枢神经系统中NMDA受体的调节。
