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Ca2+-induced Ca2+ release in chromaffin cells seen from inside the ER with targeted aequorin.

Alonso MT, Barrero MJ, Michelena P, Carnicero E, Cuchillo I, García AG, García-Sancho J, Montero M, Alvarez J - J. Cell Biol. (1999)

Bottom Line: Both InsP3 and caffeine emptied completely the ER in digitonin-permeabilized cells whereas cyclic ADP-ribose had no effect.Fast confocal [Ca2+]c measurements showed that the wave of [Ca2+]c induced by 100-ms depolarizing pulses in voltage-clamped cells was delayed and reduced in intensity in ryanodine-treated cells.Our results indicate that the ER of chromaffin cells behaves mostly as a single homogeneous thapsigargin-sensitive Ca2+ pool that can release Ca2+ both via InsP3 receptors or CICR.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biología y Genética Molecular, Departamento de Bioquímica y Biología Molecular y Fisiología, Facultad de Medicina, Universidad de Valladolid y Consejo Superior de Investigaciones Científicas, E-47005 Valladolil, Spain.

ABSTRACT
The presence and physiological role of Ca2+-induced Ca2+ release (CICR) in nonmuscle excitable cells has been investigated only indirectly through measurements of cytosolic [Ca2+] ([Ca2+]c). Using targeted aequorin, we have directly monitored [Ca2+] changes inside the ER ([Ca2+]ER) in bovine adrenal chromaffin cells. Ca2+ entry induced by cell depolarization triggered a transient Ca2+ release from the ER that was highly dependent on [Ca2+]ER and sensitized by low concentrations of caffeine. Caffeine-induced Ca2+ release was quantal in nature due to modulation by [Ca2+]ER. Whereas caffeine released essentially all the Ca2+ from the ER, inositol 1,4, 5-trisphosphate (InsP3)- producing agonists released only 60-80%. Both InsP3 and caffeine emptied completely the ER in digitonin-permeabilized cells whereas cyclic ADP-ribose had no effect. Ryanodine induced permanent emptying of the Ca2+ stores in a use-dependent manner after activation by caffeine. Fast confocal [Ca2+]c measurements showed that the wave of [Ca2+]c induced by 100-ms depolarizing pulses in voltage-clamped cells was delayed and reduced in intensity in ryanodine-treated cells. Our results indicate that the ER of chromaffin cells behaves mostly as a single homogeneous thapsigargin-sensitive Ca2+ pool that can release Ca2+ both via InsP3 receptors or CICR.

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Confocal imaging  of the propagation of the  [Ca2+]c signal induced by cell  depolarization. (a) Mean image from 34 line-scan fluorescence images representing  F/F0 (ratio between fluo-3 fluorescence at a certain time and  before stimulation, an index  for [Ca2+]c) in control cells  stimulated by a 100-ms depolarizing pulse from a holding  potential of −70 mV to 10  mV (top). (b) Mean image  from 21 records of ryanodine-treated cells displayed  as in panel a. Before starting  the experiment, cells were  exposed three times to a 10  mM caffeine + 10 μM ryanodine-containing Krebs-Hepes  solution. After that, cells were  maintained in 10 μM ryanodine during the whole experiment. c–e show the distribution with the distance to the  plasma membrane of the maximum F/F0 levels (c), the rate  of rise of fluo-3 fluorescence,  measured as the slopes calculated from the first 10 ms of  the signal rising (d), and the  time (t1.1) from the initiation  of the pulse to the moment in  which a value F/F0 ≥ 1.1  (taken as an arbitrary threshold) was reached (e).
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Figure 9: Confocal imaging of the propagation of the [Ca2+]c signal induced by cell depolarization. (a) Mean image from 34 line-scan fluorescence images representing F/F0 (ratio between fluo-3 fluorescence at a certain time and before stimulation, an index for [Ca2+]c) in control cells stimulated by a 100-ms depolarizing pulse from a holding potential of −70 mV to 10 mV (top). (b) Mean image from 21 records of ryanodine-treated cells displayed as in panel a. Before starting the experiment, cells were exposed three times to a 10 mM caffeine + 10 μM ryanodine-containing Krebs-Hepes solution. After that, cells were maintained in 10 μM ryanodine during the whole experiment. c–e show the distribution with the distance to the plasma membrane of the maximum F/F0 levels (c), the rate of rise of fluo-3 fluorescence, measured as the slopes calculated from the first 10 ms of the signal rising (d), and the time (t1.1) from the initiation of the pulse to the moment in which a value F/F0 ≥ 1.1 (taken as an arbitrary threshold) was reached (e).

Mentions: Under physiological conditions, cell stimulation is triggered by short depolarizations lasting a few milliseconds. To estimate the contribution of CICR to the Ca2+ transient under these conditions, we have compared the rate of diffusion of the Ca2+ wave induced by a short (100 ms) cell depolarization both in control cells or in cells in which the Ca2+ stores had been blocked by previous treatment with caffeine and ryanodine. We combined the whole-cell patch-clamp technique with fluo-3–based microfluorimetry using a confocal microscope. Cells were line-scanned along 100-ms square depolarizing pulses from a holding potential of −70 to +10 mV. The recorded inward currents showed two typical components: a initial transient peak (INa) followed by a slow inactivating phase (ICa) (data not shown). The ryanodine treatment did not affect the total stimulated Ca2+ entry, calculated as the integral of the last 90 ms of the recorded inward current (mean ± SEM: control cells, 7.15 ± 0.42 pC [n = 34]; ryanodine-treated cells, 6.68 ± 1.03 pC [n = 21]). In spite of this, line scan images representing [Ca2+]c showed clear differences between control and ryanodine-treated cells. Fig. 9 a shows the spatiotemporal pattern of [Ca2+]c increase in control cells, codified in pseudocolor. [Ca2+]c increased first near the plasma membrane and then the Ca2+ wave propagated intracellularly. Fig. 9 b shows the results obtained in cells with the Ca2+ stores previously emptied by treatment with caffeine and ryanodine. In this case, the [Ca2+]c increase was smaller and the propagation of the Ca2+ wave delayed. Fig. 9, panels c–e detail the behavior of several parameters that quantify the phenomenon described above in terms of peak [Ca2+]c rise (Fig. 9 c), maximum rate of [Ca2+]c increase (Fig. 9 d), and time required to increase fluorescence by 10% (Fig. 9 e) at different intracellular locations. Fig. 9 c shows that the maximum fluo-3 fluorescence (indicating the maximum [Ca2+] peak) was reached near the plasma membrane. An 80% increase was found in control cells compared with only a 40% increase in ryanodine-treated cells. The fluorescence peaks were smaller as we move deep inside the cell, but the difference among control and ryanodine-treated cells was maintained. Fig. 9 d shows that the maximum rate of fluorescence increase was located near the plasma membrane and decreased steeply as we move into the cell. Again here, the rates were two to three times faster in the control cells than in the ryanodine-treated ones. Fig. 9 e shows the time required for the fluorescence to be increased by 10% at different locations. This parameter is very sensitive to the intracellular propagation of the [Ca2+]c wave. We find that the [Ca2+]c wave propagates about twice as fast in control cells than in cells treated with ryanodine. These results indicate that CICR significantly contributes to the Ca2+ signal induced by cell depolarization during a short, more physiological stimulation.


Ca2+-induced Ca2+ release in chromaffin cells seen from inside the ER with targeted aequorin.

Alonso MT, Barrero MJ, Michelena P, Carnicero E, Cuchillo I, García AG, García-Sancho J, Montero M, Alvarez J - J. Cell Biol. (1999)

Confocal imaging  of the propagation of the  [Ca2+]c signal induced by cell  depolarization. (a) Mean image from 34 line-scan fluorescence images representing  F/F0 (ratio between fluo-3 fluorescence at a certain time and  before stimulation, an index  for [Ca2+]c) in control cells  stimulated by a 100-ms depolarizing pulse from a holding  potential of −70 mV to 10  mV (top). (b) Mean image  from 21 records of ryanodine-treated cells displayed  as in panel a. Before starting  the experiment, cells were  exposed three times to a 10  mM caffeine + 10 μM ryanodine-containing Krebs-Hepes  solution. After that, cells were  maintained in 10 μM ryanodine during the whole experiment. c–e show the distribution with the distance to the  plasma membrane of the maximum F/F0 levels (c), the rate  of rise of fluo-3 fluorescence,  measured as the slopes calculated from the first 10 ms of  the signal rising (d), and the  time (t1.1) from the initiation  of the pulse to the moment in  which a value F/F0 ≥ 1.1  (taken as an arbitrary threshold) was reached (e).
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Figure 9: Confocal imaging of the propagation of the [Ca2+]c signal induced by cell depolarization. (a) Mean image from 34 line-scan fluorescence images representing F/F0 (ratio between fluo-3 fluorescence at a certain time and before stimulation, an index for [Ca2+]c) in control cells stimulated by a 100-ms depolarizing pulse from a holding potential of −70 mV to 10 mV (top). (b) Mean image from 21 records of ryanodine-treated cells displayed as in panel a. Before starting the experiment, cells were exposed three times to a 10 mM caffeine + 10 μM ryanodine-containing Krebs-Hepes solution. After that, cells were maintained in 10 μM ryanodine during the whole experiment. c–e show the distribution with the distance to the plasma membrane of the maximum F/F0 levels (c), the rate of rise of fluo-3 fluorescence, measured as the slopes calculated from the first 10 ms of the signal rising (d), and the time (t1.1) from the initiation of the pulse to the moment in which a value F/F0 ≥ 1.1 (taken as an arbitrary threshold) was reached (e).
Mentions: Under physiological conditions, cell stimulation is triggered by short depolarizations lasting a few milliseconds. To estimate the contribution of CICR to the Ca2+ transient under these conditions, we have compared the rate of diffusion of the Ca2+ wave induced by a short (100 ms) cell depolarization both in control cells or in cells in which the Ca2+ stores had been blocked by previous treatment with caffeine and ryanodine. We combined the whole-cell patch-clamp technique with fluo-3–based microfluorimetry using a confocal microscope. Cells were line-scanned along 100-ms square depolarizing pulses from a holding potential of −70 to +10 mV. The recorded inward currents showed two typical components: a initial transient peak (INa) followed by a slow inactivating phase (ICa) (data not shown). The ryanodine treatment did not affect the total stimulated Ca2+ entry, calculated as the integral of the last 90 ms of the recorded inward current (mean ± SEM: control cells, 7.15 ± 0.42 pC [n = 34]; ryanodine-treated cells, 6.68 ± 1.03 pC [n = 21]). In spite of this, line scan images representing [Ca2+]c showed clear differences between control and ryanodine-treated cells. Fig. 9 a shows the spatiotemporal pattern of [Ca2+]c increase in control cells, codified in pseudocolor. [Ca2+]c increased first near the plasma membrane and then the Ca2+ wave propagated intracellularly. Fig. 9 b shows the results obtained in cells with the Ca2+ stores previously emptied by treatment with caffeine and ryanodine. In this case, the [Ca2+]c increase was smaller and the propagation of the Ca2+ wave delayed. Fig. 9, panels c–e detail the behavior of several parameters that quantify the phenomenon described above in terms of peak [Ca2+]c rise (Fig. 9 c), maximum rate of [Ca2+]c increase (Fig. 9 d), and time required to increase fluorescence by 10% (Fig. 9 e) at different intracellular locations. Fig. 9 c shows that the maximum fluo-3 fluorescence (indicating the maximum [Ca2+] peak) was reached near the plasma membrane. An 80% increase was found in control cells compared with only a 40% increase in ryanodine-treated cells. The fluorescence peaks were smaller as we move deep inside the cell, but the difference among control and ryanodine-treated cells was maintained. Fig. 9 d shows that the maximum rate of fluorescence increase was located near the plasma membrane and decreased steeply as we move into the cell. Again here, the rates were two to three times faster in the control cells than in the ryanodine-treated ones. Fig. 9 e shows the time required for the fluorescence to be increased by 10% at different locations. This parameter is very sensitive to the intracellular propagation of the [Ca2+]c wave. We find that the [Ca2+]c wave propagates about twice as fast in control cells than in cells treated with ryanodine. These results indicate that CICR significantly contributes to the Ca2+ signal induced by cell depolarization during a short, more physiological stimulation.

Bottom Line: Both InsP3 and caffeine emptied completely the ER in digitonin-permeabilized cells whereas cyclic ADP-ribose had no effect.Fast confocal [Ca2+]c measurements showed that the wave of [Ca2+]c induced by 100-ms depolarizing pulses in voltage-clamped cells was delayed and reduced in intensity in ryanodine-treated cells.Our results indicate that the ER of chromaffin cells behaves mostly as a single homogeneous thapsigargin-sensitive Ca2+ pool that can release Ca2+ both via InsP3 receptors or CICR.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biología y Genética Molecular, Departamento de Bioquímica y Biología Molecular y Fisiología, Facultad de Medicina, Universidad de Valladolid y Consejo Superior de Investigaciones Científicas, E-47005 Valladolil, Spain.

ABSTRACT
The presence and physiological role of Ca2+-induced Ca2+ release (CICR) in nonmuscle excitable cells has been investigated only indirectly through measurements of cytosolic [Ca2+] ([Ca2+]c). Using targeted aequorin, we have directly monitored [Ca2+] changes inside the ER ([Ca2+]ER) in bovine adrenal chromaffin cells. Ca2+ entry induced by cell depolarization triggered a transient Ca2+ release from the ER that was highly dependent on [Ca2+]ER and sensitized by low concentrations of caffeine. Caffeine-induced Ca2+ release was quantal in nature due to modulation by [Ca2+]ER. Whereas caffeine released essentially all the Ca2+ from the ER, inositol 1,4, 5-trisphosphate (InsP3)- producing agonists released only 60-80%. Both InsP3 and caffeine emptied completely the ER in digitonin-permeabilized cells whereas cyclic ADP-ribose had no effect. Ryanodine induced permanent emptying of the Ca2+ stores in a use-dependent manner after activation by caffeine. Fast confocal [Ca2+]c measurements showed that the wave of [Ca2+]c induced by 100-ms depolarizing pulses in voltage-clamped cells was delayed and reduced in intensity in ryanodine-treated cells. Our results indicate that the ER of chromaffin cells behaves mostly as a single homogeneous thapsigargin-sensitive Ca2+ pool that can release Ca2+ both via InsP3 receptors or CICR.

Show MeSH
Related in: MedlinePlus