title: Feedback activation of phospholipase C via intracellular mobilization and store-operated influx of Ca2+ in insulin-secreting beta-cells.
abstract: 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 beta-cells. Evanescent wave microscopy was used to record PLC activity using green fluorescent protein (GFP)-tagged PIP2/IP3-binding pleckstrin homology domain from PLCdelta1, and the cytoplasmic Ca2+ concentration ([Ca2+]i) was simultaneously measured using the indicator Fura Red. Stimulation of MIN6 beta-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 beta-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.
authors.affiliation: Department of Medical Cell Biology, Uppsala University, Biomedical Centre, Box 571, SE-75123 Uppsala, Sweden.
authors.forename: Sophia
authors.initials: S
authors.lastname: Thore
authors.forename: Oleg
authors.initials: O
authors.lastname: Dyachok
authors.forename: Erik
authors.initials: E
authors.lastname: Gylfe
authors.forename: Anders
authors.initials: A
authors.lastname: Tengholm
mesh_terms: D000818:Animals; D001896:Boron Compounds / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D002478:Cells, Cultured; D003981:Diazoxide / Q000378:metabolism; D004789:Enzyme Activation; D025461:Feedback, Physiological; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007328:Insulin / Q000378:metabolism*; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D007811:Lanthanum / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods; D054803:Phospholipase C delta; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism*
chemical_list: D001896:Boron Compounds; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D007328:Insulin; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D007811:Lanthanum; D015544:Inositol 1,4,5-Trisphosphate; C109986:2-aminoethoxydiphenyl borate; D010738:Type C Phospholipases; D054803:Phospholipase C delta; C518025:Plcd1 protein, mouse; D000252:Calcium-Transporting ATPases; D003981:Diazoxide; D002118:Calcium