Feedback activation of phospholipase C via intracellular mobilization and store-operated influx of Ca 2+ in insulin-secreting β-cells [Elektronisk resurs]

• Phospholipase C (PLC) regulates various cellular processes by catalyzing the formation of inositol-1,4,5-trisphosphate (IP 3 ) and diacylglycerol from phosphatidylinositol-4,5-bisphosphate (PIP 2 ). Here, we have investigated the influence of Ca 2+ 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 PIP 2 /IP 3 -binding pleckstrin homology domain from PLCδ1, and the cytoplasmic Ca 2+ concentration ([Ca 2+ ] 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 Ca 2+ or in the presence of the non-selective Ca 2+ channel inhibitor La 3+ . The Ca 2+ -dependent sustained phase of PLC activity did not require voltage-gated Ca 2+ influx, as hyperpolarization with diazoxide or direct Ca 2+ 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 Ca 2+ stores with the sarco(endo)plasmic reticulum Ca 2+ -ATPase inhibitors thapsigargin or cyclopiazonic acid abolished Ca 2+ mobilization in response to carbachol, and strongly suppressed the PLC activation in Ca 2+ -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 [Ca 2+ ] i spikes, reflecting protein kinase A-mediated activation of Ca 2+ -induced Ca 2+ release via IP 3 receptors. These [Ca 2+ ] 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 Ca 2+ 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. 

Ämnesord

Medical and Health Sciences  (hsv)

Basic Medicine  (hsv)

Cell and Molecular Biology  (hsv)

Medicin och hälsovetenskap  (hsv)

Medicinska grundvetenskaper  (hsv)

Cell- och molekylärbiologi  (hsv)

MEDICINE  (svep)

Morphology, cell biology, pathology  (svep)

Cell biology  (svep)

MEDICIN  (svep)

Morfologi, cellbiologi, patologi  (svep)

Cellbiologi  (svep)

medicinsk cellbiologi  (uu)

Medical Cell Biology  (uu)

Indexterm och SAB-rubrik

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