Lukas W, Jones K A
Glaxo Institute for Molecular Biology, Geneva, Switzerland.
Neuroscience. 1994 Jul;61(2):307-16. doi: 10.1016/0306-4522(94)90233-x.
Calbindin and the more recently identified protein calretinin are structurally related calcium-binding proteins having a broad distribution in the brain. Recent evidence supports a neuroprotective role for calbindin in regulating calcium homeostasis during periods of heightened Ca2+ influx. It is not known if calretinin might have a similar function. We investigated if calretinin-containing neurons have a survival advantage in rat neocortical cultures treated with a calcium ionophore or excitatory amino acids. Neuronal cultures were challenged with the calcium ionophore A23187 at different concentrations to produce a broad range of cell death. Cell loss was quantified for both the calretinin immunopositive and the calretinin immunonegative populations of neurons. We found that 3 h after exposure to 2 microM A23187 there was a 48% loss of the calretinin immunonegative population of neurons whereas the calretinin immunopositive set of neurons was reduced by only 18%. Calretinin positive neurons were still relatively spared after treatment with 3 microM A23187. The ionophore had no cytotoxic effect when calcium ions were removed from the extracellular medium. We also studied glutamate excitotoxicity by treating the neuronal cultures with the excitatory amino acids glutamate, N-methyl-D-aspartate or kainate for 5 min and examining survival three hours later. We found again that calretinin-containing neurons were relatively spared after exposure to the excitatory amino acids; at doses of N-methyl-D-aspartate and kainate that produced a 32-40% loss of calretinin immunonegative neurons, only 2-10% of calretinin immunopositive neurons died. Similar results were obtained for glutamate. These results demonstrate that neurons containing calretinin are better able to survive disturbances in calcium homeostasis than cells not containing this calcium-binding protein. The fact that this effect was observed with ionophore treatment, as well as excitatory amino acids, suggests that neither the density nor distribution of glutamate receptors on the different cell types was a factor in determining selective vulnerability. We hypothesize that the neuroprotective effect of calretinin is due to the buffering capacities of the protein in a manner analogous to that suggested for calbindin.
钙结合蛋白和最近发现的钙视网膜蛋白是结构相关的钙结合蛋白，在大脑中分布广泛。最近的证据支持钙结合蛋白在钙流入增加期间调节钙稳态中具有神经保护作用。尚不清楚钙视网膜蛋白是否可能具有类似功能。我们研究了在用钙离子载体或兴奋性氨基酸处理的大鼠新皮质培养物中，含钙视网膜蛋白的神经元是否具有生存优势。用不同浓度的钙离子载体A23187刺激神经元培养物，以产生广泛的细胞死亡。对钙视网膜蛋白免疫阳性和免疫阴性神经元群体的细胞损失进行定量。我们发现，暴露于2 microM A23187 3小时后，钙视网膜蛋白免疫阴性神经元群体损失了48%，而钙视网膜蛋白免疫阳性神经元组仅减少了18%。用3 microM A23187处理后，钙视网膜蛋白阳性神经元仍然相对幸免。当从细胞外培养基中去除钙离子时，离子载体没有细胞毒性作用。我们还通过用兴奋性氨基酸谷氨酸、N-甲基-D-天冬氨酸或红藻氨酸处理神经元培养物5分钟并在3小时后检查存活率来研究谷氨酸兴奋性毒性。我们再次发现，暴露于兴奋性氨基酸后，含钙视网膜蛋白的神经元相对幸免；在N-甲基-D-天冬氨酸和红藻氨酸剂量下，钙视网膜蛋白免疫阴性神经元损失了32-40%，只有2-10%的钙视网膜蛋白免疫阳性神经元死亡。谷氨酸也得到了类似的结果。这些结果表明，与不含这种钙结合蛋白的细胞相比，含钙视网膜蛋白的神经元在钙稳态紊乱中更能存活。在用离子载体处理以及兴奋性氨基酸处理时都观察到了这种效应，这一事实表明，不同细胞类型上谷氨酸受体的密度和分布都不是决定选择性易损性的因素。我们假设，钙视网膜蛋白的神经保护作用是由于该蛋白的缓冲能力，其方式类似于钙结合蛋白的情况。
