Feedback activation of phospholipase C via intracellular mobilization and store-operated influx of Ca2+ in insulin-secreting β-cells

Autor: Anders Tengholm, Oleg Dyachok, Erik Gylfe, Sophia Thore
Rok vydání: 2005
Předmět:
Isoenzymes/genetics/*metabolism
Signal Transduction/*physiology
Calcium Channels/metabolism
Cell- och molekylärbiologi
Insulin-Secreting Cells/cytology/*metabolism
Lanthanum/metabolism
Stimulation
Inositol 1
4
5-Trisphosphate
Recombinant Fusion Proteins/genetics/metabolism
Mice
chemistry.chemical_compound
Insulin-Secreting Cells
Insulin
Cells
Cultured
Feedback
Physiological
Diazoxide/metabolism
Calcium/*metabolism
Hyperpolarization (biology)
Calcium Channel Blockers
Cell biology
Isoenzymes
Ca(2+)-Transporting ATPase/antagonists & inhibitors/metabolism
Inositol 1
5-Trisphosphate/metabolism
Boron Compounds/metabolism
Phospholipase C/genetics/*metabolism
Cyclopiazonic acid
Intracellular
Signal Transduction
Boron Compounds
Microscopy
Fluorescence/methods
Thapsigargin
Recombinant Fusion Proteins
Green Fluorescent Proteins
Calcium-Transporting ATPases
Biology
Insulin/*metabolism
BAPTA
Lanthanum
Green Fluorescent Proteins/genetics/metabolism
Feedback
Biochemical
Animals
Research Support
Non-U.S. Gov't
Diacylglycerol kinase
Phospholipase C
Diazoxide
Cell Biology
Calcium Channel Blockers/metabolism
Enzyme Activation
Microscopy
Fluorescence
chemistry
Type C Phospholipases
Calcium
Cells
Cultured
Calcium Channels
Phospholipase C delta
Cell and Molecular Biology
Zdroj: Journal of Cell Science. 118:4463-4471
ISSN: 1477-9137
0021-9533
DOI: 10.1242/jcs.02577
Popis: 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.
Databáze: OpenAIRE
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