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Producción Científica, We have used an aequorin chimera targeted to the membrane of the secretory granules to monitor the free [Ca2+] inside them in neurosecretory PC12 cells. More than 95% of the probe was located in a compartment with an homogeneous [Ca2+] around 40 M. Cell stimulation with either ATP, caffeine or high-K+ depolarization increased cytosolic [Ca2+] and decreased secretory granule [Ca2+] ([Ca2+]SG). Inositol-(1,4,5)- trisphosphate, cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate were all ineffective to release Ca2+ from the granules. Changes in cytosolic [Na+] (0–140 mM) or [Ca2+] (0–10 M) did not modify either ([Ca2+]SG). Instead, [Ca2+]SG was highly sensitive to changes in the pH gradient between the cytosol and the granules. Both carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and nigericin, as well as cytosolic acidification, reversibly decreased [Ca2+]SG, while cytosolic alcalinization reversibly increased [Ca2+]SG. These results are consistent with the operation of a H+/Ca2+ antiporter in the vesicular membrane. This antiporter could also mediate the effects of ATP, caffeine and high-K+ on [Ca2+]SG, because all of them induced a transient cytosolic acidification. The FCCP-induced decrease in [Ca2+]SG was reversible in 10–15 min even in the absence of cytosolic Ca2+ or ATP, suggesting that most of the calcium content of the vesicles is bound to a slowly exchanging Ca2+ buffer. This large store buffers [Ca2+]SG changes in the long-term but allows highly dynamic free [Ca2+]SG changes to occur in seconds or minutes.

Cellular Ca 2+signaling results from a complex interplay among a variety of Ca 2+ fluxes going across the plasma membrane and across the membranes of several organelles, together with the buffering effect of large numbers of Ca 2+-binding sites distributed along the cell architecture. Endoplasmic and sarcoplasmic reticulum, mitochondria and even nucleus have all been involved in cellular Ca 2+ signaling, and the mechanisms for Ca 2+ uptake and release from these organelles are well known. In neuroendocrine cells, the secretory granules also constitute a very important Ca 2+-storing organelle, and the possible role of the stored Ca 2+ as a trigger for secretion has attracted considerable attention. However, this possibility is frequently overlooked, and the main reason for that is that there is still considerable uncertainty on the main questions related with granular Ca 2+ dynamics, e.g., the free granular [Ca 2+], the physical state of the stored Ca 2+ or the mechanisms for Ca 2+ accumulation and release from the granules. This review will give a critical overview of the present state of knowledge and the main conflicting points on secretory granule Ca 2+ homeostasis in neuroendocrine cells. © 2011 Elsevier Ltd., This work was supported by grants from Ministerio de Educación y Ciencia (BFU2008-01871) and from Junta de Castilla y León (GR105).