Kawaminami M, Okazaki K, Uchida S, Marumoto N, Takehara K, Kurusu S, Hashimoto I, Walker A M
Veterinary Physiology, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, 034, Aomori, Japan,
Endocrine. 1996 Aug;5(1):9-14. doi: 10.1007/BF02738650.
Annexin 5 is expressed by rat anterior pituitary cells and a depolarizing stimulus results in increased extracellular display and, depending on local calcium concentrations, potential release into the extracellular environment. In order to further investigate the role of annexin 5 in anterior pituitary function, we have examined the intracellular distribution by immunocytochemistry and the effects of annexin 5 on the release of a major secretory product, prolactin. Prolactin was chosen because we could easily monitor effects on basal release and effects on the immediate and sustained phases of thyroid stimulating hormone releasing hormone (TRH)-stimulated release. Immunocytochemical localization of annexin 5 showed staining of the majority of anterior pituitary cells. Labeling was predominantly on the nuclear envelope and plasma membrane. For the chosen secretory product, prolactin, annexin 5 was found in most, but not all prolactin positive cells. When recombinant annexin 5 (50 ng/mL) was added to a 3 h static culture incubation of rat anterior pituitary cells, prolactin release was inhibited by about 30% (p<0.05). A lower dose had a reduced effect and higher doses had no further inhibitory effect, indicating that the effect was specific to annexin 5 and not a nonspecific toxic effect of some contaminant in the preparation. This interpretation was further strengthened in a time-course experiment demonstrating that when TRH and annexin 5 were added together, there was no effect of annexin 5 on the amount of prolactin released. After a 3 h preincubation in annexin 5, however, prolactin release, in response to TRH, was suppressed by about 30% in both the acute and sustained phases. These data suggest that annexin 5 may be a local regulator of release in the anterior pituitary, but a slow onset effect on both phases of TRH-stimulated release suggests that this is not an effect at the plasma membrane such as local extracellular calcium depletion by plasma membrane-bound annexin 5.
膜联蛋白5由大鼠垂体前叶细胞表达，去极化刺激导致细胞外展示增加，并根据局部钙浓度，可能释放到细胞外环境中。为了进一步研究膜联蛋白5在垂体前叶功能中的作用，我们通过免疫细胞化学检查了其细胞内分布以及膜联蛋白5对主要分泌产物催乳素释放的影响。选择催乳素是因为我们可以轻松监测其对基础释放的影响以及对促甲状腺激素释放激素（TRH）刺激释放的即刻和持续阶段的影响。膜联蛋白5的免疫细胞化学定位显示大多数垂体前叶细胞有染色。标记主要位于核膜和质膜上。对于所选的分泌产物催乳素，在大多数但并非所有催乳素阳性细胞中都发现了膜联蛋白5。当将重组膜联蛋白5（50 ng/mL）添加到大鼠垂体前叶细胞的3小时静态培养孵育中时，催乳素释放受到约30%的抑制（p<0.05）。较低剂量的作用较小，较高剂量没有进一步的抑制作用，表明该作用对膜联蛋白5具有特异性，而不是制剂中某些污染物的非特异性毒性作用。在一项时间进程实验中进一步证实了这一解释，该实验表明当TRH和膜联蛋白5一起添加时，膜联蛋白5对释放的催乳素量没有影响。然而，在膜联蛋白5中预孵育3小时后，对TRH的反应中，催乳素释放在急性和持续阶段均被抑制约30%。这些数据表明膜联蛋白5可能是垂体前叶释放的局部调节因子，但对TRH刺激释放的两个阶段起效缓慢表明这不是质膜上的作用，例如质膜结合的膜联蛋白5导致局部细胞外钙耗竭。
