Calcium dynamics in catecholamine-containing secretory vesicles

We have used an aequorin chimera targeted to the membrane of the secretory granules to monitor the free Ca(2+)] inside them in neurosecretory PC12 cells. More than 95% of the probe was located in a compartment with an homogeneous Ca(2+)] around 40 microM. Cell stimulation with either ATP, caffeine or high-K(+) depolarization increased cytosolic Ca(2+)] and decreased secretory granule Ca(2+)] (Ca(2+)](SG)). Inositol-(1,4,5)-trisphosphate, cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate were all ineffective to release Ca(2+) from the granules. Changes in cytosolic Na(+)] (0-140 mM) or Ca(2+)] (0-10 microM) did not modify either (Ca(2+)](SG)). Instead, Ca(2+)](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 Ca(2+)](SG), while cytosolic alcalinization reversibly increased Ca(2+)](SG). These results are consistent with the operation of a H(+)/Ca(2+) antiporter in the vesicular membrane. This antiporter could also mediate the effects of ATP, caffeine and high-K(+) on Ca(2+)](SG), because all of them induced a transient cytosolic acidification. The FCCP-induced decrease in Ca(2+)](SG) was reversible in 10-15 min even in the absence of cytosolic Ca(2+) or ATP, suggesting that most of the calcium content of the vesicles is bound to a slowly exchanging Ca(2+) buffer. This large store buffers Ca(2+)](SG) changes in the long-term but allows highly dynamic free Ca(2+)](SG) changes to occur in seconds or minutes.