title: Oscillations of phospholipase C activity triggered by depolarization and Ca2+ influx in insulin-secreting cells.
abstract: Phospholipase C (PLC) is a ubiquitous enzyme involved in the regulation of a variety of cellular processes. Its dependence on Ca2+ is well recognized, but it is not known how PLC activity is affected by physiological variations of the cytoplasmic Ca2+ concentration ([Ca2+](i)). Here, we applied evanescent wave microscopy to monitor PLC activity in parallel with [Ca2+](i) in individual insulin-secreting INS-1 cells using the phosphatidylinositol 4,5-bisphosphate- and inositol 1,4,5-trisphosphate-binding pleckstrin homology domain from PLCdelta(1) fused to green fluorescent protein (PH(PLCdelta1)-GFP) and the Ca2+ indicator fura red. In resting cells, PH(PLCdelta1)-GFP was located predominantly at the plasma membrane. Activation of PLC by muscarinic or purinergic receptor stimulation resulted in PH(PLCdelta1)-GFP translocation from the plasma membrane to the cytoplasm, detected as a decrease in evanescent wave-excited PH(PLCdelta1)-GFP fluorescence. Using this translocation as a measure of PLC activity, we found that depolarization by raising extracellular [K+] triggered activation of the enzyme. This effect could be attributed both to a rise of [Ca2+](i) and to depolarization per se, because some translocation persisted during depolarization in a Ca2+-deficient medium containing the Ca2+ chelator EGTA. Moreover, oscillations of [Ca2+](i) resulting from depolarization with Ca2+ influx evoked concentration-dependent periodic activation of PLC. We conclude that PLC activity is under tight dynamic control of [Ca2+](i). In insulin-secreting beta-cells, this mechanism provides a link between Ca2+ influx and release from intracellular stores that may be important in the regulation of insulin secretion.
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: Anders
authors.initials: A
authors.lastname: Tengholm
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D001692:Biological Transport; D001798:Blood Proteins / Q000378:metabolism; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002217:Carbachol / Q000737:chemistry; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009991:Oscillometry; D010750:Phosphoproteins / Q000378:metabolism; D011188:Potassium / Q000737:chemistry; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D051381:Rats; D013997:Time Factors; D014162:Transfection; D010738:Type C Phospholipases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001798:Blood Proteins; D002614:Chelating Agents; D007328:Insulin; D008164:Luminescent Proteins; D010750:Phosphoproteins; D020033:Protein Isoforms; C039077:platelet protein P47; D049452:Green Fluorescent Proteins; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D002217:Carbachol; D010738:Type C Phospholipases; D011188:Potassium; D002118:Calcium