Calcium puffs are generic InsP3-activated elementary calcium signals and are downregulated by prolonged hormonal stimulation to inhibit cellular calcium responses
Stephen C. Tovey1,*,
Patrick de Smet2,*,
Peter Lipp1,
,
David Thomas1,
Kenneth W. Young3,
Ludwig Missiaen2,
Humbert De Smedt2,
Jan B. Parys2,
Michael J. Berridge1,
Jan Thuring4,
Andrew Holmes4 and
Martin D. Bootman1,5
1 Laboratory of Molecular Signalling, The Babraham Institute, Babraham, Cambridge, CB2 4AT, UK
2 Laboratory of Physiology, K.U. Leuven Campus Gasthuisberg O/N, Herestraat 49, B-3000 Leuven, Belgium
3 Department of Cell Physiology and Pharmacology, Medical Sciences Building, University of Leicester, University Road, Leicester, LE1 9HN, UK
4 Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK
5 Department of Zoology, University of Cambridge, Downing Street, Cambridge, CB2 3EJ, UK
* These authors contributed equally to this study
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Fig. 1. InsP3R expression and localization. (A) The histograms illustrate the relative abundances of the three InsP3R isoforms in the cell types listed. The data were obtained by analysing the density of the immunoreactive bands at  260 kDa. The data show means±s.e.m. of three blots. (B) Typical confocal images (z depth <1 µm) of (left) HeLa cells stained for type 1 InsP3Rs, (middle) HUVEC cells stained for type 2 InsP3Rs and (right) 16HBE14o- cells stained for type 3 InsP3Rs. In most of the cell types, a reticular distribution of staining could be observed but, in 16HBE14o- cells in particular, the ER seemed to be considerably fragmented after fixation. (C) Intensity profiles of immunofluorescence from the nuclear envelope to the plasma membrane. These profiles were obtained by sampling the immunofluorescence intensity of the major InsP3R isoform in each cell type across a line one pixel wide running from the nuclear envelope to the plasma membrane.
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Fig. 2. Ca2+ puff activity during local and global signalling. All of the cell types analysed in the present study displayed Ca2+ puffs when treated with appropriate agonist concentrations. The aim of this figure is to show some of the different modes in which Ca2+ puff activity can be observed in these cells. (A) Rapid Ca2+ puff activity in a 16HBE14o- cell stimulated with 5 µM ATP. Several Ca2+ puff sites were active before and during the onset of the Ca2+ wave. White circles on the inset cell image show the locations of the active puff sites, and the Ca2+ signals recorded at these regions are depicted by the correspondingly numbered traces. (B) A HeLa cell stimulated with 1 µM histamine and in which a single Ca2+ puff sites was responsible for triggering a regenerative Ca2+ wave. The black trace represents the Ca2+ signal observed at the puff site and the red trace depicts the global Ca2+ signal observed by averaging fluo-3 fluorescence across the whole cell. (C) Ca2+ puffs firing at three different sites within a single carbachol-stimulated SH-SY5Y cell (1 µM carbachol). Coloured circles on the inset cell image show the locations of the active puff sites, and the Ca2+ signals recorded at these regions are depicted by the correspondingly coloured traces. This example illustrates the observation that, sometimes, the Ca2+ puff sites appear to fire in synchrony, whereas, at other times, they do not. (D) Ca2+ puffs in a NIH-3T3 cell stimulated with 1 µM ATP. Ca2+ puffs were observed at the regions marked by the blue, green and red circles on the inset cell image, and the correspondingly coloured traces indicate the Ca2+ changes observed at these sites. Ca2+ puffs were observed during the initial latency before the first Ca2+ oscillation and also on the rising phases of subsequent Ca2+ oscillations.
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Fig. 3. Intracellular position of pacemaker Ca2+ puffs. (A) The histograms show the distribution of Ca2+ puffs relative to the nuclear envelope for each of the cell types investigated. (B) The average nuclear distances of the pacemaker Ca2+ puff sites are plotted against cell diameter, showing a roughly linear correlation for all the cell types except RBL-2H3 cells. The data are shown as mean±s.e.m. The dashed line (drawn by eye) indicates the trend for the pacemaker Ca2+ puff sites to be more distant from the nuclear boundary as the cell size increases. The cell size was estimated by measuring the diameter of cells along randomly chosen lines that crossed the cell body and nucleus.
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Fig. 4. Ca2+ puffs in 16HBE14o- and SH-SY5Y cells. (A) High-frequency non-regenerative Ca2+ puffs in a 16HBE14o- cell. The top two traces depict Ca2+ puffs firing at the sites marked by the correspondingly coloured circles in the inset cell image. The ticks beneath the second trace denote the occurrence of a Ca2+ puff at either of the sites. The average frequency of events in this recording was 0.8 Hz. Despite this high frequency, there was only a modest effect on the global Ca2+ concentration (bottom trace). (B) The relatively slow elevation of the intracellular Ca2+ concentration observed in some SH-SY5Y cells. Although the cell shown had two active pacemaker Ca2+ puff sites, a regenerative Ca2+ rise was not observed. Instead, there was a steady shallow Ca2+ increase. The dashed lines in the cell images represent the positions of the nuclei.
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Fig. 5. Downregulation of InsP3Rs in SH-SY5Y cells. (A) The effect of prolonged incubation with 1 mM carbachol on the expression level of (i) InsP3R type 1 and (ii) InsP3R type 3 in SH-SY5Y cells. Typical examples of the immunoband at 261 kDa and 248 kDa for types 1 and 3 InsP3Rs are shown above the histograms. The immunoreactivity is expressed as a percentage of that in untreated cells. The data are expressed as mean±s.e.m. (n=3). All data were significantly different from control (P<0.05). (B) The functional loss of InsP3Rs in cells incubated with 1 mM carbachol for 6 hours. To provide a more reliable estimate of the global Ca2+ response, we used ratiometric imaging of Fura2 rather than single wavelength confocal recording. The data are expressed as percentage (mean±s.e.m.) of the amplitude of the Ca2+ fluorescence signal in untreated cells. The number of experiments (n) is indicated in the bars.
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Fig. 6. Changes in Ca2+ puff characteristics in SH-SY5Y cells following prolonged agonist stimulation. (A-D) The effects of incubating SH-SY5Y cells with 1 mM carbachol for 4 hours on the characteristics of Ca2+ puffs. The Gaussian curves were calculated using Microcal Origin (Northampton, USA).
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Fig. 7. Downregulation of InsP3Rs and changes in Ca2+ puff characteristics in HeLa cells following prolonged agonist stimulation. (A) The effect of prolonged incubation with 1 mM histamine on the expression level of (i) IP3R type 1 and (ii) IP3R type 3 in HeLa cells. The immunoreactivity is expressed as a percentage of that in untreated cells. The data are expressed as mean±s.e.m. (n=3). (B) The functional loss of InsP3Rs in cells incubated with 1 mM histamine for 6 hours assessed using ratiometric imaging of Fura2. The data are expressed as percentage (mean±s.e.m.) of the amplitude of the Ca2+ fluorescence signal in untreated cells. The number of experiments (n) is indicated in the bars. (C,D) The change in the Ca2+ puff characteristics following incubation of HeLa cells with 1 mM histamine for 4 hours.
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Fig. 8. Inhibition of Ca2+ wave initiation following prolonged agonist stimulation of HeLa cells. Typical responses of single HeLa cells to 20 µM InsP3BM either before (A) or after (B) a 4-hour incubation with 1 mM histamine. (A) An example in which the pacemaking Ca2+ puff activity was able to drive the cell towards the threshold for triggering a regenerative global Ca2+ wave. (B) The failure of the Ca2+ puffs to trigger a regenerative response in a cell pre-incubated with 1 mM histamine. The cell shown in (B) was chosen because it displayed three active pacemaking Ca2+ puff sites and was one of the most active cells following the histamine preincubation. However, despite the considerable activity of the pacemaking Ca2+ puff sites, they caused only modest changes in the global Ca2+ concentration. The cells shown in this figure were from the same passage and are typical of the responses from three independent experiments. Coloured circles on the inset cell images depict the positions of the pacemaking Ca2+ puffs.
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