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 μM in both cell types, about half the values previously found in chromaffin cells. Inhibition of SERCA-type Ca2+ pumps with thapsigargin largely blocked Ca2+ uptake by the granules in Ca2+-depleted permeabilized cells, and the same effect was obtained when the perfusion medium lacked ATP. Consistently, the SERCA-type Ca2+ pump inhibitor benzohydroquinone 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 Ca2+ 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, supporting the hypothesis that secretory granule Ca2+ may be released during cell stimulation and contribute to secretion.
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Abbreviations
- BHQ:
-
tert-Butyl benzohydroquinone
- [Ca2+]SG :
-
Secretory granule [Ca2+]
- DMPP:
-
1,1-Dimethyl-4-phenyl-piperazinium iodide
- InsP3 :
-
Inositol 1,4,5-trisphosphate
- SERCA:
-
Sarco endoplasmic reticulum Ca2+ ATPase
- VAMP:
-
Vesicle-associated membrane protein
- VAMP-mutaeq:
-
VAMP-mutated aequorin chimera
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Acknowledgments
This work was supported by grants from Ministerio de Educación y Ciencia (BFU2008-01871) and from Junta de Castilla y León (VA103A08 and GR105). J.S. had a FPI (Formación de Personal Investigador) fellowship from the Spanish Ministerio de Educación y Ciencia (MEC).
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A commentary to this article can be found at doi:10.1007/s10571-010-9611-z.
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SantoDomingo, J., Fonteriz, R.I., Lobatón, C.D. et al. Ca2+ Dynamics in the Secretory Vesicles of Neurosecretory PC12 and INS1 Cells. Cell Mol Neurobiol 30, 1267–1274 (2010). https://doi.org/10.1007/s10571-010-9572-2
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DOI: https://doi.org/10.1007/s10571-010-9572-2