Miyata S, Nakai S, Kiyohara T, Hatton G I
Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan.
J Neurocytol. 2000 Jan;29(1):5-17. doi: 10.1023/a:1007180328597.
Ca(2+) binding proteins (CaBPs), calbindin-D(28k) (calbindin) and calretinin, are thought to contribute to the regulation of intracellular Ca(2+) in many neuronal populations and perhaps more importantly, signal functional modulation in neuronal activity. In the present experiments, light microscopic immunohistochemistry revealed that the immunoreactivity of calbindin and calretinin was contained in varicose axons in the posterior pituitary. The dual labeling study with confocal microscopy demonstrated that calbindin immunoreactivity was present in the terminals of both oxytocin (OXT) and arginine-vasopressin (AVP) neurons. However, calretinin immunoreactivity was exclusively seen in the OXT terminals. Moreover, the dual labeling study showed that most calretinin-positive terminals contained calbindin immunoreactivity, demonstrating the colocalization of calbindin and calretinin in the same OXT nerve terminals. By electron microscopy, calbindin and calretinin immunoreactivities were seen in the neurosecretory axons and nerve terminals. These immunoreactive nerve terminals were seen to contain more clear microvesicles than dense-core neurosecretory granules. This immunoelectron microscopic observation suggests that both calbindin and calretinin localize preferentially in the active zone of the nerve terminals, which usually face the perivascular space around fenestrated capillaries. In spite of similar localization of calbindin and calretinin within the posterior pituitary, Western blot analysis showed some differences between the two CaBPs. Calbindin was present mostly in the soluble fraction with little in the insoluble fraction, but a substantial portion of calretinin was present in both the insoluble and soluble fractions. Moreover, dehydration induced by drinking 2% NaCl solution and deprivation of drinking water increased calretinin levels in the posterior pituitary as compared with control, but the calbindin level was not changed. The present findings demonstrate that calbindin and calretinin colocalize in the active zones of OXT nerve terminals, but only calretinin is upregulated with dehydration, suggesting different physiological role of calbindin and calretinin in the nerve terminals.
钙离子结合蛋白（CaBPs），即钙结合蛋白-D(28k)（钙结合蛋白）和钙视网膜蛋白，被认为在许多神经元群体中有助于调节细胞内钙离子，或许更重要的是，它们在神经元活动中发挥信号功能调节作用。在本实验中，光学显微镜免疫组织化学显示，钙结合蛋白和钙视网膜蛋白的免疫反应性存在于垂体后叶的曲张轴突中。共聚焦显微镜双标研究表明，催产素（OXT）神经元和精氨酸加压素（AVP）神经元的终末均存在钙结合蛋白免疫反应性。然而，钙视网膜蛋白免疫反应性仅见于OXT终末。此外，双标研究表明，大多数钙视网膜蛋白阳性终末含有钙结合蛋白免疫反应性，表明钙结合蛋白和钙视网膜蛋白在同一OXT神经终末中共定位。通过电子显微镜观察，在神经分泌轴突和神经终末中可见钙结合蛋白和钙视网膜蛋白免疫反应性。这些免疫反应性神经终末所含的清亮微泡比致密核心神经分泌颗粒更多。这种免疫电子显微镜观察结果表明，钙结合蛋白和钙视网膜蛋白均优先定位于神经终末的活性区，该活性区通常面向有孔毛细血管周围的血管周隙。尽管钙结合蛋白和钙视网膜蛋白在垂体后叶内的定位相似，但蛋白质免疫印迹分析显示这两种钙离子结合蛋白之间存在一些差异。钙结合蛋白主要存在于可溶性部分，不溶性部分含量很少，而相当一部分钙视网膜蛋白同时存在于不溶性和可溶性部分。此外，饮用2%氯化钠溶液诱导的脱水和禁水与对照组相比，垂体后叶中钙视网膜蛋白水平升高，但钙结合蛋白水平未改变。本研究结果表明，钙结合蛋白和钙视网膜蛋白在OXT神经终末的活性区共定位，但只有钙视网膜蛋白在脱水时上调，提示钙结合蛋白和钙视网膜蛋白在神经终末具有不同的生理作用。
