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First published online 13 September 2005
doi: 10.1242/jcs.02577

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

¹ Department of Medical Cell Biology, Uppsala University, Biomedical Centre, Box 571, SE-75123 Uppsala, Sweden
² Department of Biophysics, National T. Shevchenko University of Kiev, Kiev, Ukraine

View larger version (23K): [in a new window]   Fig. 1. Ca2+-dependent, biphasic activation of PLC in insulin-secreting cells. (A) MIN6 cells were transiently transfected with PHPLC-GFP and visualized 24 hours later with evanescent wave microscopy under basal conditions and after stimulation with 100 µM carbachol at the time points i and ii indicated in B. Bars 5 µm. (B) Time-course of PHPLC-GFP translocation in the cell shown in A after stimulation with 100 µM carbachol. (C) Time-course of PHPLC-GFP translocation in response to 100 µM carbachol in Ca2+-deficient medium containing 2 mM EGTA. (D) Effect of Ca2+ removal with addition of 2 mM EGTA on the steady-state PHPLC-GFP fluorescence during stimulation with 100 µM carbachol. (E) Time-course of PHPLC-GFP translocation in response to 100 µM carbachol in the presence of 0.5 mM La3+. The inset shows the entire experiment, including the rise of PHPLC-GFP fluorescence that occurs upon addition of La3+. The fluorescence was normalized against the level prior to carbachol stimulation. (F) Mean±s.e.m. for the effects of Ca2+ removal and La3+ addition on PHPLC-GFP fluorescence. The peak amplitude was defined as the maximal change from initial fluorescence and the plateau amplitude was calculated from the off-response upon washout of carbachol. **P<0.001; *P<0.01.

View larger version (23K): [in a new window]   Fig. 2. PLC activity is maintained by Ca2+ influx through store-operated channels. Evanescent wave microscopy recording of PHPLC-GFP-expressing MIN6 cells during sustained PLC activity triggered by 100 µM carbachol. (A) Effect of 250 µM diazoxide. (B) Effect of 10 µM nifedipine and 100 µM 2-APB. (C) Effect of 50 µM SKF96365. (D) Effect of 100 µM carbachol and 100 µM 2-APB in cells expressing membrane-targeted GFP. (E) Mean±s.e.m. for the effects of diazoxide, nifedipine, 2-APB and SKF96365 on the steady-state PHPLC-GFP fluorescence during stimulation with carbachol. *P<0.01; **P<0.001. (F) Effect of carbachol when added after 100 µM 2-APB. (G) Effect of store-operated Ca2+ influx triggered by 100 µM CPA on PHPLC-GFP fluorescence.

View larger version (23K): [in a new window]   Fig. 3. PLC activation involves positive feedback from intracellular Ca2+ mobilization. (A) Simultaneous evanescent wave microscopy fluorescence recording of cytoplasmic Ca2+ concentration ([Ca2+]i) with Fura Red (red trace) and PLC activity with PHPLC-GFP (green trace) in a MIN6 cell stimulated with 100 µM carbachol in Ca2+-deficient medium (0 Ca2+/2 mM EGTA). The Fura Red trace has been inverted to show [Ca2+]i increases as upward deflections. (B) Carbachol-induced PHPLC-GFP translocation and [Ca2+]i response in a cell preincubated for 40 minutes with 1 mM BAPTA acetoxymethyl ester and maintained in Ca2+-deficient medium from 5 minutes prior to stimulation. (C) PHPLC-GFP translocation and [Ca2+]i response in a cell maintained in Ca2+-deficient medium containing 100 µM cyclopiazonic acid. (D,E) Mean±s.e.m. for peak PHPLC-GFP translocation and [Ca2+]i responses. *P<0.01; **P<0.001.

View larger version (25K): [in a new window]   Fig. 4. Ca2+ mobilization activates PLC in primary mouse pancreatic ß-cells. (A) Mouse pancreatic ß-cells transiently transfected with PHPLC-YFP and loaded with Fura Red were transferred to medium containing 20 mM glucose, 250 µM diazoxide, 50 µM verapamil and 100 nM glucagon. PLC activity (PHPLC-YFP fluorescence, green trace) and [Ca2+]i response (red trace) were recorded simultaneously with an image pair every 0.7 seconds. The Fura Red trace has been inverted to show [Ca2+]i increases as upward deflections. (B) Time expansions of the shaded regions in A showing that the [Ca2+]i responses precede PLC activation. The recording is representative for six cells from five independent experiments.

View larger version (16K): [in a new window]   Fig. 5. Model for Ca2+ regulation of receptor-triggered PLC activity in insulin-secreting cells. Agonist (A) stimulation of G-protein-coupled receptors (GPCR) leads to partial activation of PLC via the Gq family of heterotrimeric G proteins. The resulting hydrolysis of membrane PIP2 leads to formation of diacylglycerol (DAG) and sufficient amounts of IP3 to mobilize Ca2+ via IP3 receptors (IP3R) in the endoplasmic reticulum (ER). The elevation of [Ca2+]i further activates PLC to stimulate formation of more IP3, which leads to further elevation of [Ca2+]i, etc. The drop of Ca2+ concentration inside the ER triggers activation of store-operated channels (SOC) in the plasma membrane. This store-operated Ca2+ entry acts to increase [Ca2+]i and to stimulate PLC activity during prolonged receptor stimulation.