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Feedback activation of phospholipase C via intracellular mobilization and store-operated influx of Ca2+ in insulin-secreting β-cells
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
2005 (English)In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 118, no Pt 19, p. 4463-4471Article in journal (Refereed) Published
Abstract [en]

Phospholipase C (PLC) regulates various cellular processes by catalyzing the formation of inositol-1,4,5-trisphosphate (IP3) and diacylglycerol from phosphatidylinositol-4,5-bisphosphate (PIP2). Here, we have investigated the influence of Ca2+ on receptor-triggered PLC activity in individual insulin-secreting β-cells. Evanescent wave microscopy was used to record PLC activity using green fluorescent protein (GFP)-tagged PIP2/IP3-binding pleckstrin homology domain from PLCδ1, and the cytoplasmic Ca2+ concentration ([Ca2+]i) was simultaneously measured using the indicator Fura Red. Stimulation of MIN6 β-cells with the muscarinic-receptor agonist carbachol induced rapid and sustained PLC activation. By contrast, only transient activation was observed after stimulation in the absence of extracellular Ca2+ or in the presence of the non-selective Ca2+ channel inhibitor La3+. The Ca2+-dependent sustained phase of PLC activity did not require voltage-gated Ca2+ influx, as hyperpolarization with diazoxide or direct Ca2+ channel blockade with nifedipine had no effect. Instead, the sustained PLC activity was markedly suppressed by the store-operated channel inhibitors 2-APB and SKF96365. Depletion of intracellular Ca2+ stores with the sarco(endo)plasmic reticulum Ca2+-ATPase inhibitors thapsigargin or cyclopiazonic acid abolished Ca2+ mobilization in response to carbachol, and strongly suppressed the PLC activation in Ca2+-deficient medium. Analogous suppressions were observed after loading cells with the Ca2+ chelator BAPTA. Stimulation of primary mouse pancreatic β-cells with glucagon elicited pronounced [Ca2+]i spikes, reflecting protein kinase A-mediated activation of Ca2+-induced Ca2+ release via IP3 receptors. These [Ca2+]i spikes were found to evoke rapid and transient activation of PLC. Our data indicate that receptor-triggered PLC activity is enhanced by positive feedback from Ca2+ entering the cytoplasm from intracellular stores and via store-operated channels in the plasma membrane. Such amplification of receptor signalling should be important in the regulation of insulin secretion by hormones and neurotransmitters.

Place, publisher, year, edition, pages
2005. Vol. 118, no Pt 19, p. 4463-4471
Keywords [en]
Animals, Boron Compounds/metabolism, Ca(2+)-Transporting ATPase/antagonists & inhibitors/metabolism, Calcium/*metabolism, Calcium Channel Blockers/metabolism, Calcium Channels/metabolism, Cells; Cultured, Diazoxide/metabolism, Enzyme Activation, Feedback; Biochemical, Green Fluorescent Proteins/genetics/metabolism, Inositol 1;4;5-Trisphosphate/metabolism, Insulin/*metabolism, Insulin-Secreting Cells/cytology/*metabolism, Isoenzymes/genetics/*metabolism, Lanthanum/metabolism, Mice, Microscopy; Fluorescence/methods, Phospholipase C/genetics/*metabolism, Recombinant Fusion Proteins/genetics/metabolism, Research Support; Non-U.S. Gov't, Signal Transduction/*physiology
National Category
Cell and Molecular Biology
Research subject
Medical Cell Biology
Identifiers
URN: urn:nbn:se:uu:diva-79778DOI: 10.1242/jcs.02577PubMedID: 16159958OAI: oai:DiVA.org:uu-79778DiVA, id: diva2:107691
Available from: 2009-01-29 Created: 2008-11-21 Last updated: 2018-01-13Bibliographically approved
In thesis
1. Regulation of Phospholipase C and Plasma Membrane Phosphatidylinositol 4,5-bisphosphate in Insulin-Secreting Cells
Open this publication in new window or tab >>Regulation of Phospholipase C and Plasma Membrane Phosphatidylinositol 4,5-bisphosphate in Insulin-Secreting Cells
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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-δ1 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 β-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 ADPβS. 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 Iβ. 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.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2006. p. 46
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 126
Keywords
Cell biology, PLC, PIP2, insulin-secreting cell, Ca2+, evanescent wave microscopy, phosphoinositide kinase, ATP/ADP ratio, Cellbiologi
National Category
Cell Biology
Identifiers
urn:nbn:se:uu:diva-6677 (URN)91-554-6502-1 (ISBN)
Public defence
2006-04-27, Room B21, Biomedicinskt centrum, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2006-04-06 Created: 2006-04-06 Last updated: 2009-10-14Bibliographically approved

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