Santodomingo Jaime, Vay Laura, Camacho Marcial, Hernández-Sanmiguel Esther, Fonteriz Rosalba I, Lobatón Carmen D, Montero Mayte, Moreno Alfredo, Alvarez Javier
Departamento de Bioquímica y Biología Molecular y Fisiología, Facultad de Medicina, Universidad de Valladolid and Consejo Superior de Investigaciones Científicas (CSIC), Ramón y Cajal, 7, E-47005 Valladolid, Spain.
Eur J Neurosci. 2008 Oct;28(7):1265-74. doi: 10.1111/j.1460-9568.2008.06440.x.
The secretory granules constitute one of the less well-known compartments in terms of Ca2+ dynamics. They contain large amounts of total Ca2+, but the free intragranular [Ca2+] ([Ca2+]SG), the mechanisms for Ca2+ uptake and release from the granules and their physiological significance regarding exocytosis are still matters of debate. We used in the present work an aequorin chimera targeted to the granules to investigate [Ca2+]SG homeostasis in bovine adrenal chromaffin cells. We found that most of the intracellular aequorin chimera is present in a compartment with 50-100 microM Ca2+. Ca2+ accumulation into this compartment takes place mainly through an ATP-dependent mechanism, namely, a thapsigargin-sensitive Ca2+-ATPase. In addition, fast Ca2+ release was observed in permeabilized cells after addition of inositol 1,4,5-trisphosphate (InsP3) or caffeine, suggesting the presence of InsP3 and ryanodine receptors in the vesicular membrane. Stimulation of intact cells with the InsP3-producing agonist histamine or with caffeine also induced Ca2+ release from the vesicles, whereas acetylcholine or high-[K+] depolarization induced biphasic changes in vesicular[Ca2+], suggesting heterogeneous responses of different vesicle populations, some of them releasing and some taking up Ca2+during stimulation. In conclusion, our data show that chromaffin cell secretory granules have the machinery required for rapid uptake and release of Ca2+, and this strongly supports the hypothesis that granular Ca2+ may contribute to its own secretion.
就钙离子动力学而言，分泌颗粒是了解较少的区室之一。它们含有大量的总钙离子，但颗粒内游离钙离子浓度（[Ca2+]SG）、钙离子摄入和从颗粒中释放的机制及其关于胞吐作用的生理意义仍存在争议。在本研究中，我们使用靶向颗粒的水母发光蛋白嵌合体来研究牛肾上腺嗜铬细胞中[Ca2+]SG的稳态。我们发现，细胞内大部分水母发光蛋白嵌合体存在于钙离子浓度为50 - 100微摩尔的区室中。钙离子主要通过一种依赖ATP的机制，即毒胡萝卜素敏感的钙离子ATP酶，积累到该区室中。此外，在加入肌醇1,4,5 - 三磷酸（InsP3）或咖啡因后，在透化细胞中观察到快速的钙离子释放，这表明囊泡膜中存在InsP3和兰尼碱受体。用产生InsP3的激动剂组胺或咖啡因刺激完整细胞也会诱导囊泡中的钙离子释放，而乙酰胆碱或高钾去极化会诱导囊泡[Ca2+]的双相变化，这表明不同囊泡群体有不同反应，其中一些在刺激过程中释放钙离子，一些则摄取钙离子。总之，我们的数据表明嗜铬细胞分泌颗粒具有快速摄取和释放钙离子所需的机制，这有力地支持了颗粒钙离子可能有助于其自身分泌的假说。
