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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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Activation of  CICR by the Ca2+ entry elicited by high K+-induced cell  depolarization. The ER was  refilled by perfusing medium containing 1 mM Ca2+.  Then, standard medium containing 70 mM KCl was perfused as indicated. In c, 10  μM CPA was added as indicated with or without high  K+ medium. In this panel,  the [Ca2+]ER scale has been  normalized as percentage of  the maximum [Ca2+]ER level  in order to facilitate comparison. In the presence of CPA  and high K+ medium the initial rate of [Ca2+]ER decrease is much higher that  with CPA alone, but once  [Ca2+]ER is below 80% of the  initial level, the rates of release in both cases turn similar and can be nearly superimposed. In d, 1 mM caffeine  was also added as indicated.  Other details are as in Fig. 1.
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Figure 5: Activation of CICR by the Ca2+ entry elicited by high K+-induced cell depolarization. The ER was refilled by perfusing medium containing 1 mM Ca2+. Then, standard medium containing 70 mM KCl was perfused as indicated. In c, 10 μM CPA was added as indicated with or without high K+ medium. In this panel, the [Ca2+]ER scale has been normalized as percentage of the maximum [Ca2+]ER level in order to facilitate comparison. In the presence of CPA and high K+ medium the initial rate of [Ca2+]ER decrease is much higher that with CPA alone, but once [Ca2+]ER is below 80% of the initial level, the rates of release in both cases turn similar and can be nearly superimposed. In d, 1 mM caffeine was also added as indicated. Other details are as in Fig. 1.

Mentions: The next step in this study was to investigate the presence of CICR activated by Ca2+ entry through the plasma membrane Ca2+ channels. Depolarization with high K+ medium or by stimulation with nicotinic acetylcholine agonists such as 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP), produced large [Ca2+]c peaks (Núñez et al., 1995), that were not significantly modified by previous Ca2+ depletion of the ER with caffeine or with the ER Ca2+ pump inhibitor thapsigargin (see below). Therefore, the possible contribution of CICR to these [Ca2+]c peaks cannot be estimated from conventional [Ca2+]c studies, and direct measurement of [Ca2+]ER becomes essential. Fig. 5 a shows that 10-s pulses of depolarization with high K+ medium induced a transient Ca2+ release from the ER, which could be triggered repetitively by consecutive pulses. The [Ca2+]ER decrease was of 60–100 μM (10–15% of the steady-state [Ca2+]ER). Therefore, in spite of the large increase in [Ca2+]c, the activation of CICR produced a much smaller [Ca2+]ER decrease than treatment with caffeine. Increasing the duration of the high K+ pulse increased the magnitude of Ca2+ release little. Fig. 5 b illustrates the effect of depolarization with longer (1 min) high K+ pulses. In this case, the first high K+ pulse produced the same effect as in Fig. 5 a, and then [Ca2+]ER increased more rapidly, probably as a result of the prolonged stimulation of the ER Ca2+ pump by the sustained high [Ca2+]c levels. This led to a new [Ca2+]ER steady-state at ∼800–900 μM. After that, subsequent K+ pulses induced somewhat larger [Ca2+]ER decreases of 150–200 μM, but corresponding still to only 20% of the steady-state [Ca2+]ER. To investigate if increased Ca2+ pumping could be responsible for the incomplete Ca2+ release, the effect of K+ depolarization was tested in the presence of the ER Ca2+ pump inhibitor ciclopiazonic acid (CPA). Fig. 5 c shows that CPA itself induces a slow Ca2+ release from the ER, and that simultaneous addition of high K+ medium induced a fast initial Ca2+ release of ∼20% of the [Ca2+]ER, followed by a slower release at a rate comparable to that induced by CPA alone. This suggests that CICR induced by a maximal K+ depolarization is able to produce only a decrease of ∼20% of the [Ca2+]ER, even in the absence of Ca2+ pumping. CICR, however, was potentiated by simultaneous addition of a low caffeine concentration. Fig. 5 d shows that addition of 1 mM caffeine produced little effect by itself, but strongly potentiated the effect of K+ depolarization, that was now able to release rapidly ∼50% of the stored Ca2+. Finally, CICR could also be triggered in a more physiological way using an agonist for the nicotinic acetylcholine receptor. Addition of DMPP induced a rapid and partial Ca2+ release from the ER, very similar to that shown above for K+ depolarization, and which was also potentiated by low concentrations of caffeine (data not shown).


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)

Activation of  CICR by the Ca2+ entry elicited by high K+-induced cell  depolarization. The ER was  refilled by perfusing medium containing 1 mM Ca2+.  Then, standard medium containing 70 mM KCl was perfused as indicated. In c, 10  μM CPA was added as indicated with or without high  K+ medium. In this panel,  the [Ca2+]ER scale has been  normalized as percentage of  the maximum [Ca2+]ER level  in order to facilitate comparison. In the presence of CPA  and high K+ medium the initial rate of [Ca2+]ER decrease is much higher that  with CPA alone, but once  [Ca2+]ER is below 80% of the  initial level, the rates of release in both cases turn similar and can be nearly superimposed. In d, 1 mM caffeine  was also added as indicated.  Other details are as in Fig. 1.
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Related In: Results  -  Collection

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Figure 5: Activation of CICR by the Ca2+ entry elicited by high K+-induced cell depolarization. The ER was refilled by perfusing medium containing 1 mM Ca2+. Then, standard medium containing 70 mM KCl was perfused as indicated. In c, 10 μM CPA was added as indicated with or without high K+ medium. In this panel, the [Ca2+]ER scale has been normalized as percentage of the maximum [Ca2+]ER level in order to facilitate comparison. In the presence of CPA and high K+ medium the initial rate of [Ca2+]ER decrease is much higher that with CPA alone, but once [Ca2+]ER is below 80% of the initial level, the rates of release in both cases turn similar and can be nearly superimposed. In d, 1 mM caffeine was also added as indicated. Other details are as in Fig. 1.
Mentions: The next step in this study was to investigate the presence of CICR activated by Ca2+ entry through the plasma membrane Ca2+ channels. Depolarization with high K+ medium or by stimulation with nicotinic acetylcholine agonists such as 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP), produced large [Ca2+]c peaks (Núñez et al., 1995), that were not significantly modified by previous Ca2+ depletion of the ER with caffeine or with the ER Ca2+ pump inhibitor thapsigargin (see below). Therefore, the possible contribution of CICR to these [Ca2+]c peaks cannot be estimated from conventional [Ca2+]c studies, and direct measurement of [Ca2+]ER becomes essential. Fig. 5 a shows that 10-s pulses of depolarization with high K+ medium induced a transient Ca2+ release from the ER, which could be triggered repetitively by consecutive pulses. The [Ca2+]ER decrease was of 60–100 μM (10–15% of the steady-state [Ca2+]ER). Therefore, in spite of the large increase in [Ca2+]c, the activation of CICR produced a much smaller [Ca2+]ER decrease than treatment with caffeine. Increasing the duration of the high K+ pulse increased the magnitude of Ca2+ release little. Fig. 5 b illustrates the effect of depolarization with longer (1 min) high K+ pulses. In this case, the first high K+ pulse produced the same effect as in Fig. 5 a, and then [Ca2+]ER increased more rapidly, probably as a result of the prolonged stimulation of the ER Ca2+ pump by the sustained high [Ca2+]c levels. This led to a new [Ca2+]ER steady-state at ∼800–900 μM. After that, subsequent K+ pulses induced somewhat larger [Ca2+]ER decreases of 150–200 μM, but corresponding still to only 20% of the steady-state [Ca2+]ER. To investigate if increased Ca2+ pumping could be responsible for the incomplete Ca2+ release, the effect of K+ depolarization was tested in the presence of the ER Ca2+ pump inhibitor ciclopiazonic acid (CPA). Fig. 5 c shows that CPA itself induces a slow Ca2+ release from the ER, and that simultaneous addition of high K+ medium induced a fast initial Ca2+ release of ∼20% of the [Ca2+]ER, followed by a slower release at a rate comparable to that induced by CPA alone. This suggests that CICR induced by a maximal K+ depolarization is able to produce only a decrease of ∼20% of the [Ca2+]ER, even in the absence of Ca2+ pumping. CICR, however, was potentiated by simultaneous addition of a low caffeine concentration. Fig. 5 d shows that addition of 1 mM caffeine produced little effect by itself, but strongly potentiated the effect of K+ depolarization, that was now able to release rapidly ∼50% of the stored Ca2+. Finally, CICR could also be triggered in a more physiological way using an agonist for the nicotinic acetylcholine receptor. Addition of DMPP induced a rapid and partial Ca2+ release from the ER, very similar to that shown above for K+ depolarization, and which was also potentiated by low concentrations of caffeine (data not shown).

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