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Rapid Turnover of Phosphatidylinositol-4,5-Bisphosphate in Insulin-Secreting Cells Mediated by Ca²⁺ and the ATP-to-ADP Ratio

From the Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden

Address correspondence and reprint requests to Anders Tengholm, Department of Medical Cell Biology, Uppsala University, Biomedical Centre, Box 571, SE-75123 Uppsala, Sweden. E-mail: anders.tengholm{at}mcb.uu.se

Abbreviations: ADPßS, adenosine-5'-O-2-thiodiphosphate; [Ca²⁺]_i, cytoplasmic Ca²⁺ concentration; GFP, green fluorescent protein; IP_3, inositol-1,4,5-trisphosphate; K_ATP channel, ATP-sensitive K⁺ channel; PH, pleckstrin homology; PI 4-kinase, phosphatidylinositol 4-kinase; PIP, phosphatidylinositol-4-phosphate; PIP₂, phosphatidylinositol-4,5-bisphosphate; PLC, phospholipase C

Phosphatidylinositol-4,5-bisphosphate (PIP₂) is important for a variety of cellular processes as a precursor for second messengers and by regulating ion channels, the cytoskeleton, and vesicle traffic in many types of cells, including insulin-secreting ß-cells. Here, we applied evanescent wave microscopy and the PIP₂-binding pleckstrin homology domain from phospholipase C (PLC)- fused to the green fluorescent protein to characterize the regulation of plasma membrane PIP₂ in individual insulin-secreting MIN6 ß-cells. Elevation of the glucose concentration from 3 to 11 mmol/l evoked antisynchronous oscillations of [PIP₂] and cytoplasmic Ca²⁺concentration, consistent with PLC being periodically activated by the voltage-dependent Ca²⁺ influx. The effect of adenine nucleotides on [PIP₂] was studied in cells permeabilized with -toxin. ATP dose- dependently stimulated PIP₂ synthesis with half-maximal effect at 300 µmol/l. Omission of the nucleotide resulted in rapid loss of PIP₂ with t_1/2 < 40 s. ADP also stimulated PIP₂ formation, but this effect reflected local ATP formation and was prevented by the adenylate kinase inhibitor diadenosine-pentaphosphate. The ATP-induced PIP₂ synthesis was counteracted by the ADP analog adenosine-5'-O-2-thiodiphosphate. We conclude that plasma membrane PIP₂ is dynamically regulated by intracellular Ca²⁺ and the ATP-to-ADP ratio in insulin-secreting cells. The rapid turnover allows maintenance of PIP₂ levels while generating second messengers of critical importance for insulin secretion.

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