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oai:uvadoc.uva.es:10324/59732024-12-16T12:05:43Zcom_10324_1133com_10324_931com_10324_894col_10324_1209

Calcium dynamics in catecholamine-containing secretory vesicles Moreno Díaz-Calderón, Alfredo Domínguez Lobatón, María Carmen Santo Domingo Mayoral, Jaime Vay, Laura Hernández San Miguel, Esther Rizzuto, Rosario Montero Zoccola, María Teresa Álvarez Martín, Javier Calcio en el organismo 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. 2014-09-16T08:47:12Z 2014-09-16T08:47:12Z 2005 info:eu-repo/semantics/article Cell Calcium, 2005, vol. 37, p. 555-564 0143-4160 http://uvadoc.uva.es/handle/10324/5973 10.1016/j.ceca.2005.02.002 555 564 Cell Calcium 37 eng Attribution-NonCommercial-NoDerivatives 4.0 International info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by-nc-nd/4.0/ application/pdf Elsevier Ltd. https://uvadoc.uva.es/bitstream/10324/5973/6/ALVAREZ%2015-%20Calcium%20dynamics%20in%20catecholamine.pdf.jpg Hispana TEXT http://creativecommons.org/licenses/by-nc-nd/4.0/ UVaDOC. Repositorio Documental de la Universidad de Valladolid http://uvadoc.uva.es/handle/10324/5973