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First published online November 23, 2005
doi: 10.1242/10.1242/jcs.02663

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Carboxyamidotriazole-induced inhibition of mitochondrial calcium import blocks capacitative calcium entry and cell proliferation in HEK-293 cells

¹ Department of Pharmacology and Physiology, University of Rochester, 601 Elmwood Avenue, Rochester, NY 14642, USA
² Department of Neuroscience, Medical College of Ohio, 3036 Arlington Avenue, Toledo, OH 43614, USA
³ INSERM, EMI 0228, IFR118, Université des Sciences et Technologies de Lille 1, Bât. SN3, 59655 Villeneuve d'Ascq CEDEX, France
⁴ INSERM, U442, IFR46, Université Paris-Sud, Bât.443, 91405 Orsay CEDEX, France

View larger version (34K): [in a new window]   Fig. 1. 2-APB- and CAI-evoked inhibition of CCE in HEK-293 cells. Increases in [Ca2+]i are expressed as ratios of 340/380 nm fluorescence signals. Cells were incubated in the absence of Ca2+ but with thapsigargin (TG, 1 µM), which depletes intracellular Ca2+ stores and activates CCE. CCE was estimated as the difference between [Ca2+]i increase in the presence and absence of TG (Basal). 2-APB and CAI were added 45 seconds and 300 seconds, respectively before 2 mM Ca2+. Individual traces show the responses to TG, CCE activation and the inhibitory effects of 2-APB (A) and CAI (C). Mean F340/F380 ratios from a series of experiments in the presence of increasing concentrations of 2-APB (B) and CAI (D). Values are the mean±s.e.m.

View larger version (20K): [in a new window]   Fig. 2. Time-dependent effect of CAI on CCE inhibition. Thapsigargin (1 µM) and Ca2+ (2 mM) were added to a suspension of HEK-293 cells. (A) CAI (10 µM) was added either 45 seconds (thin solid trace) or 300 seconds (dashed line) before Ca2+ and the CCE increase was compared with that in control cells (thick solid trace) and the [Ca2+]i increase in the absence of Ca2+ store depletion (basal, not shown). (B) Mean results from a series of experiments similar to those shown in A. Values are the mean±s.e.m.; n=39, 3, 10 and 4 for control, CAI 300 seconds, CAI 45 seconds and basal, respectively.

View larger version (14K): [in a new window]   Fig. 3. CAI-induced inhibition of store-operated inward calcium currents. (A) HEK-293 cells were pre-incubated with 5 µM CAI (thin trace) or not (CTRL, thick trace). Gaps in the current traces correspond to the time taken to apply voltage ramps to determine current/potential relationships. The addition of 5 µM CAI (horizontal line) did not reduce the amplitude of the plateau phase of the inward current recorded in the control conditions. Whole-cell currents were measured using 250 millisecond voltage steps from a holding potential of 0 to -80 mV delivered every 2 seconds. (B) Mean results from a series of experiments similar to those described in A. Values are the mean±s.e.m.; n=5.

View larger version (67K): [in a new window]   Fig. 4. Effect of CAI and FCCP on mitochondrial inner-membrane potential (m). (A) Following complete Ca2+ store depletion by perfusion with 10 µM TG in nominal Ca2+-containing solution for about 5 minutes, 5 mM Ca2+ was added to the perfusate (arrowhead) evoking an increase in [Ca2+]m in control cells loaded with 2 µM Rhod-2/AM. This increase was greatly attenuated in loaded cells co-treated with 10 µM TG and 20 µM FCCP. (B) Application of CAI (10 µM) or FCCP (20 µM) inhibited m compared with vehicle-treated (0.1% DMSO) controls as determined by JC-1 dye red-to-green fluorescence ratio. (C) Confocal images of JC-1-loaded (2 µg/ml) HEK-293 cells pretreated for 20 minutes with vehicle, CAI or FCCP. Bars, 10 µm.

View larger version (21K): [in a new window]   Fig. 5. CAI depolarizes the mitochondrial inner-membrane potential in a concentration-dependent manner. (A) Digital images of TMRE fluorescence in a single HEK-293 cell 60 seconds prior to (i) and 10 seconds following (ii) FCCP application (indicated by bar). (iii) Line traces of intensity changes from mitochondria indicated by white chevrons in (i). Note the spontaneous changes in m and loss of fluorescence on depolarization when TMRE was loaded at low concentration. (B) Conversely, following high concentration loading of TMRE (i), FCCP application induced an increase in fluorescence in a small cluster of HEK-293 cells (ii); a response to depolarization and unquenching of the fluorophore as dye redistributed to the cytosol. (iii) Averaged intensity trace of rises in fluorescence measured in individual cells. (C) Average fluorescence intensity changes of high concentration TMRE-loaded cells treated with vehicle only (filled symbols) or treated with 10 µM CAI (open symbols). Cell suspensions were analyzed using a multi-well fluorescence plate reader. The break in the records indicates application of the drug. Bars, 10 µm.

View larger version (28K): [in a new window]   Fig. 6. Effect of CAI treatment on [Ca2+]m dynamics in digitonin-permeabilized HEK-293 cells. (A) Mean Rhod-2/MitoTracker Green ratio fluorescence changes for suspensions of permeabilized HEK cells pre-treated with 30 µM Ca2+. Cells were treated at time 0 with vehicle (closed circles) or 10 µM CAI (open circles) and fluorescence changes were monitored by multi-well fluorescence plate reader. (B) Changes in [Ca2+]m in permeabilized HEK cells in response to intracellular saline solutions containing specific set levels of [Ca2+] (3-3000 µM) measured as in Fig. 3. (C) Relationship between the concentration of Ca2+ added and the mean change in mitochondrial fluorescence in permeabilized cells. Mitochondrial fluorescence was measured 300 seconds after adding Ca2+. , 10 µM CAI-treated cells; , vehicle-treated cells. (D) Effect of pre-treatment with CAI on change in [Ca2+]m induced by adding 30 µM Ca2+. Representative traces are shown, demonstrating an attenuation and/or loss of mitochondrial fluorescence compared with the control. (E) Representative digital fluorescence images (a-e) of HEK cells loaded with 2 µM mixture of Rhod-2, Rhod-FF and Rhod-5N dyes (1:1:1). Cells were permeabilized with 10 µM digitonin for 3 minutes in nominal Ca2+ and then challenged (arrow) with the saline solution containing 300 µM free Ca2+. The trace is the mean time-dependent change in fluorescence measured from individual puncta (see insets). Ca2+-containing solutions were applied locally (about 50-100 µm from the field of view) using a pressure-driven perfusion system. Bars, 10 µm.

View larger version (14K): [in a new window]   Fig. 7. Inhibition of [3H]thymidine incorporation by CAI and 2-APB in HEK-293 cells. HEK-293 cells were incubated for 24 hours in the presence of 10% FCS and for a further 24 hours in the presence of various concentrations of CAI (A) and 2-APB (B). Results are expressed as a percentage of [3H]thymidine incorporation in the absence of CAI and 2-APB in both conditions. The result for each experiment is the mean of four samples and each point on the plot is the mean±s.e.m. of three or four independent experiments.

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