Biochemical and Biophysical Research Communications
Calcium-dependent translocation of cytosolic phospholipase A2 in pancreatic β-cells
Section snippets
Methods
Materials. MIN6 cells were provided by Professor J.-I. Miyazaki (University of Osaka, Japan). Tissue culture reagents were obtained from Invitrogen (Paisley, UK) and general purpose laboratory reagents were purchased from Sigma (Poole, UK). The anti-cPLA2 antibody was a gift from the Genetics Institute (Cambridge, MA, USA). Alexa 488 goat anti-rabbit IgG was purchased from Molecular Probes (Oregon, USA).
MIN6 cell culture. MIN6 cells were maintained in culture on tissue culture grade plasticware
Stimulus-induced changes in intracellular Ca2+ in MIN6 cell clusters
Estimated basal cytosolic Ca2+ (ratio 340/380 nm) was 0.76±0.14 (n=69 cells) in unstimulated (2 mM glucose) MIN6 cells, and 98% of cells responded to a KCl-evoked (20 mM) depolarisation with a rapid elevation in intracellular Ca2+ (58/59 cells in eight separate experiments; Fig. 1). Similarly, the sulphonylurea tolbutamide (100 μM) evoked a rapid (within 1 min) increase in [Ca2+]i in 97% of cells examined (33/34 cells from four separate experiments; Fig. 1). The mean basal-to-peak changes in
Discussion
In many cell types, cPLA2 activity is regulated through Ca2+-dependent translocation to membrane compartments enriched in the phosphatidylcholine substrate and it is usually localised to the cytosol in unstimulated cells, as the name suggests (reviewed in [1], [8]). However, the current confocal immunofluorescence studies indicate that in unstimulated MIN6 β-cells cPLA2 is distributed throughout the cell and is not confined to the cytosolic compartment. Our previously published confocal
Acknowledgements
We thank Professor J.-I. Miyazaki for provision of the MIN6 cells and the Genetics Institute for the cPLA2 antibody. This work was funded by Grants from Diabetes UK: RD97/0001525 to P.M.J. and S.J.P., and RD97/0001453 and RD01/0002216 to PES.
References (20)
- et al.
Structure, function and regulation of the Ca2+-sensitive phospholipase A2
FEBS Lett.
(1997) - et al.
Phospholipase A2 expression in human and rodent insulin-secreting cells
Mol. Cell. Endocrinol.
(1995) Properties and regulation of cytosolic phospholipase A2
J. Biol. Chem.
(1997)- et al.
The synchronization of Ca2+-signals within insulin-secreting pseudoislets is not dependent on gap junctions
Cell Calcium
(2000) - et al.
Depolarizing stimuli reduce Ca2+/calmodulin-dependent protein kinase II activity in islets of Langerhans
Biochem. Biophys. Res. Commun.
(2000) - et al.
Nuclear import of hepatic glucokinase depends upon glucokinase regulatory protein, whereas export is due to a nuclear export signal sequence in glucokinase
J. Biol. Chem.
(1999) - et al.
Identification and localisation of a type IV cytosolic PLA2 in rat pancreatic β cells
J. Mol. Endocrinol.
(1996) - et al.
Identification of cytosolic PLA2 isoforms in human and rat pancreatic islets and insulin-secreting β-cell lines
Endocrinology
(1996) - et al.
Mass spectrometric identification and quantification of arachidonate-containing phospholipids in pancreatic islets: prominence of plasmenylethanolamine molecular species
Biochemistry
(1993) - et al.
A key role for β-cell cytosolic phospholipase A2 in the maintenance of insulin stores, but not in the initiation of insulin secretion
Diabetes
(2002)
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