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J. Biol. Chem., Vol. 283, Issue 33, 22383-22389, August 15, 2008

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Intravesicular Calcium Release Mediates the Motion and Exocytosis of Secretory Organelles

A STUDY WITH ADRENAL CHROMAFFIN CELLS^*

Marcial Camacho¹, José D. Machado², Javier Alvarez, and Ricardo Borges

From the Unidad de Farmacología, Facultad de Medicina, Universidad de La Laguna Tenerife, E-38071 La Laguna, Spain and the Instituto de Biología y Genética Molecular, Valladolid E-47005, Spain

Secretory vesicles of sympathetic neurons and chromaffin granules maintain a pH gradient toward the cytosol (pH 5.5 versus 7.2) promoted by the V-ATPase activity. This gradient of pH is also responsible for the accumulation of amines and Ca²⁺ because their transporters use H⁺ as the counter ion. We have recently shown that alkalinization of secretory vesicles slowed down exocytosis, whereas acidification caused the opposite effect. In this paper, we measure the alkalinization of vesicular pH, caused by the V-ATPase inhibitor bafilomycin A1, by total internal reflection fluorescence microscopy in cells overexpressing the enhanced green fluorescent protein-labeled synaptobrevin (VAMP2-EGFP) protein. The disruption of the vesicular gradient of pH caused the leak of Ca²⁺, measured with fura-2. Fluorimetric measurements, using the dye Oregon green BAPTA-2, showed that bafilomycin directly released Ca²⁺ from freshly isolated vesicles. The Ca²⁺ released from vesicles to the cytosol dramatically increased the granule motion of chromaffin- or PC12-derived granules and triggered exocytosis (measured by amperometry). We conclude that the gradient of pH of secretory vesicles might be involved in the homeostatic regulation of cytosolic Ca²⁺ and in two of the major functions of secretory cells, vesicle motion and exocytosis.

Received for publication, January 23, 2008 , and in revised form, June 9, 2008.

^* This work was supported in part by Spanish Ministerio de Educación y Ciencia Grants BFU2004-08038 (to R. B.) and BFU2005-05464 (to J. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Movies S1 and S2 and Fig. S1.

¹ Recipient of a Formación Personal Investigador fellowship.

² Recipient of a "Juan de La Cierva" contract. To whom correspondence should be addressed: Unidad de Farmacología., Facultad de Medicina, Universidad de La Laguna Tenerife, E-38071 La Laguna, Spain. Tel.: 34-922-319346; Fax: 34-922-655995; E-mail: jdmacha{at}ull.es.

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