ORIGINAL RESEARCH Ca2+ Dynamics in the Secretory Vesicles of Neurosecretory PC12

Abstract We have investigated the dynamics of the free [Ca2?] inside the secretory granules of neurosecretory PC12 and INS1 cells using a low-Ca2?-affinity aequorin chimera fused to synaptobrevin-2. The steady-state secretory granule [Ca2?] ([Ca2?]SG] was around 20–40 lM in both cell types, about half the values previously found in chromaffin cells. Inhibition of SERCA-type Ca2? pumps with thapsigar-gin largely blocked Ca2? uptake by the granules in Ca2?-depleted permeabilized cells, and the same effect was obtained when the perfusion medium lacked ATP. Consis-tently, the SERCA-type Ca2? pump inhibitor benzohydro-quinone induced a rapid release of Ca2? from the granules both in intact and permeabilized cells, suggesting that the continuous activity of SERCA-type Ca2? pumps is essential to maintain the steady-state [Ca2?]SG. Both inositol 1,4, 5-trisphosphate (InsP3) and caffeine produced a rapid Ca 2? release from the granules, suggesting the presence of InsP3 and ryanodine receptors in the granules. The response to high-K? depolarization was different in both cell types, a decrease in [Ca2?]SG in PC12 cells and an increase in [Ca2?]SG in INS1 cells. The difference may rely on the heterogeneous response of different vesicle populations in each cell type. Finally, increasing the glucose concentration triggered a decrease in [Ca2?]SG in INS1 cells. In conclusion, our data show that the secretory granules of PC12 and INS1 cells take up Ca2? through SERCA-type Ca2? pumps and can release it through InsP3 and ryanodine receptors, sup-porting the hypothesis that secretory granule Ca2? may be released during cell stimulation and contribute to secretion