Calcium-dependent translocation of cytosolic phospholipase A2 in pancreatic β-cells

https://doi.org/10.1016/S0006-291X(02)02969-8Get rights and content

Abstract

Cytosolic phospholipase A2(cPLA2), an enzyme responsible for the generation of arachidonic acid, is located in the cytosolic compartment in most tissues and it translocates to membrane compartments when activated. We found that cPLA2 distribution in pancreatic β-cells is different from that of most other mammalian cells: it is evenly distributed throughout the β-cell, in both cytoplasmic and nuclear compartments. Agents that increased intracellular Ca2+ in the MIN6 β-cell line also stimulated a redistribution of cPLA2 immunoreactivity such that the majority of the enzyme moved from the nucleus to the cytoplasm. The time course of events was compatible with the elevation in Ca2+ being responsible for translocation of cPLA2. These observations suggest that cPLA2 may be compartmentalised in unstimulated β-cells, perhaps to limit its access to substrate prior to elevations in intracellular Ca2+.

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)

There are more references available in the full text version of this article.

Cited by (8)

  • Expression of 25-hydroxyvitamin D<inf>3</inf>-1α-hydroxylase in pancreatic islets

    2004, Journal of Steroid Biochemistry and Molecular Biology
View all citing articles on Scopus
View full text