Regulation of Phospholipase C and Plasma Membrane Phosphatidylinositol 4,5-bisphosphate in Insulin-Secreting Cells

Abstract: The membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) is an important signaling molecule as substrate for the phospholipase C (PLC)-catalyzed formation of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol, and by directly regulating e.g. ion-channels, the cytoskeleton and vesicle trafficking in various types of cells. The present studies provide insights into the regulation of PLC activity and the plasma membrane concentration of PIP2 in individual insulin-secreting cells. Real-time monitoring of plasma membrane PIP2 was performed with evanescent wave microscopy and the PIP2/IP3-binding pleckstrin-homology-domain from PLC-?1 fused to GFP. It was demonstrated that membrane depolarization and voltage-dependent Ca2+ influx are sufficient to activate PLC. Rise of the glucose concentration triggered Ca2+-dependent activation of PLC. Simultaneous measurements of the cytoplasmic Ca2+ concentration ([Ca2+]i) demonstrated that oscillations of [Ca2+]i resulting from periodic influx induced cyclic activation of PLC. Activation of muscarinic receptors caused a biphasic PLC response with an initial peak enhanced by positive feedback by Ca2+ mobilized from intracellular stores, followed by sustained activity depending on store-operated Ca2+-entry. Activation of PLC by Ca2+ mobilized from intracellular stores was part of the Ca2+-induced Ca2+ release mechanism by which glucagon stimulates primary mouse pancreatic ?-cells. Experiments in permeabilized cells demonstrated rapid turnover of PIP2 with t1/2 ~ 16s. ATP stimulated concentration-dependent synthesis of plasma membrane PIP2, counteracted by the ADP analogue ADP?S. RT-PCR analysis identified transcripts of 10 different phosphoinositide-kinases. The ATP-stimulated PIP2 formation was mediated by type II and III PI4-kinases as well as by PIP5-kinase I?. It is concluded that the PIP2 concentration in the plasma membrane is regulated by the ATP/ADP ratio and that its hydrolysis by PLC is tightly controlled by [Ca2+]i in insulin-secreting cells.