Real-time analysis of phospholipase C activity during different patterns of receptor-induced Ca2+ responses in HEK293 cells.
[Ca(2+)](i) oscillations can either depend on oscillatory inositol-1,4,5-trisphosphate (InsP(3)) formation by phospholipase C (PLC) or rely on local feedback mechanisms involving the InsP(3) receptor. To assess the PLC activity underlying carbachol-induced [Ca(2+)](i) oscillations in single HEK293 cells, we co-imaged [Ca(2+)](i) with fluorescent fusion proteins of protein kinase C (PKC) isotypes and the PH domain of PLC-delta 1 (PLC-delta 1(PH)). The translocation of PKC alpha-YFP in single cells followed two discrete patterns. Upon maximally effective agonist concentrations, a fast association and delayed dissociation (k(on)>k(off)) was the predominant pattern. The delayed dissociation has been linked to diacylglycerol formation. Upon stimulation with submaximally effective agonist concentrations as well as during regenerative [Ca(2+)](i) waves, we mainly observed short translocations with k(on) approximately equal to k(off). Translocation time courses and efficiencies of the diacylglycerol-sensing PKC epsilon-CFP and the InsP(3)/phosphatidylinositol-4,5-bisphosphate-sensing YFP-PLC-delta 1(PH) were closely correlated. Significant PLC activity was only detectable upon strong receptor stimulation, which typically failed to trigger [Ca(2+)](i) oscillations. During [Ca(2+)](i) oscillations induced by submaximal receptor stimulation, YFP-PLC-delta 1(PH) did not translocate, whereas a fluorescent PKC epsilon fusion protein has been reported to exhibit a slow, non-oscillatory accumulation at the plasma membrane. We conclude that carbachol-induced [Ca(2+)](i) oscillations in HEK293 cells develop at low levels of presumably non-oscillatory PLC activity.
Sinnecker
Daniel
D
Institut für Pharmakologie, Freie Universität Berlin, Thielallee 67-73, 14195 Berlin, Germany.
Schaefer
Michael
M
D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D054803:Phospholipase C delta; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D051381:Rats; D011976:Receptors, Muscarinic / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism*
D007527:Isoenzymes; D011976:Receptors, Muscarinic; D011993:Recombinant Fusion Proteins; D002217:Carbachol; D011493:Protein Kinase C; D010738:Type C Phospholipases; D054803:Phospholipase C delta