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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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Regulation of caffeine-induced Ca2+ release by  [Ca2+]ER. The ER was refilled by perfusing with medium containing 1 mM Ca2+ and then 1 μM histamine or different concentrations of caffeine were added, as indicated. In the experiment  shown in c, cells were incubated with 10 μM BAPTA-AM during  aequorin reconstitution in order to load the cytosol with this  Ca2+ chelator. Other details are as in Fig. 1.
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Figure 4: Regulation of caffeine-induced Ca2+ release by [Ca2+]ER. The ER was refilled by perfusing with medium containing 1 mM Ca2+ and then 1 μM histamine or different concentrations of caffeine were added, as indicated. In the experiment shown in c, cells were incubated with 10 μM BAPTA-AM during aequorin reconstitution in order to load the cytosol with this Ca2+ chelator. Other details are as in Fig. 1.

Mentions: The experiments of Fig. 3 are consistent with previous results obtained looking at [Ca2+]c in the same cell preparation by Cheek et al., 1994a. Those experiments led the authors to suggest that there should be different compartments within the ER having different sensitivities to caffeine. This hypothesis would explain why ryanodine only empties the pool sensitive to 5 mM caffeine but leaves the rest of the pool untouched, which could be only released with a higher caffeine dose. However, there is an alternative explanation for these results, based on the regulation of caffeine-induced Ca2+ release by the lumenal [Ca2+]. In this hypothesis, the results can be explained with only one ER compartment if we assume that submaximal caffeine concentrations can only release Ca2+ until [Ca2+]ER is reduced to a certain level. The higher the caffeine concentration, the lower the [Ca2+]ER level attained. Both alternative hypotheses lead to different predictions under some experimental conditions. In particular, if we obtain half-filled stores by different procedures, e.g., by emptying them with an agonist acting via InsP3 production or by refilling the stores only halfway, the hypothesis of several compartments predicts that we should have all of them half-filled. Therefore, a submaximal dose of caffeine should still release Ca2+ from half of them. On the contrary, if the release was directly controlled by [Ca2+]ER, we would expect the effect of 5 mM caffeine to be independent of the procedure used to reach that half-filling. The experiments shown in Fig. 4 indicate that the last hypothesis is the correct one. In Fig. 4 a, half-filling was obtained by emptying the ER with histamine. After that, 5 mM caffeine had no effect. Instead, if 5 mM caffeine was added with the ER full of Ca2+, it was able to empty it exactly down to the same point. Fig. 4 b shows the effect of half-filling the ER by reducing the time of refilling. Again, the effect of 5 mM caffeine was strictly dependent on the level of [Ca2+]ER reached at the point it was added. It had no effect at half-filling, but released 50% of the pool when the Ca2+ stores were completely filled. Fig. 4 c shows a similar approach but made in cells preloaded with the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) to slow the refilling. We can observe again that the effect of 5 mM caffeine was strictly dependent on the [Ca2+]ER at the moment of addition. In addition, this experiment also shows that Ca2+ release induced by caffeine requires only resting [Ca2+]c. Fura-2 measurements performed in parallel showed that in cells loaded with BAPTA, the [Ca2+]c changes induced by caffeine were almost abolished (data not shown). This result points out also that quantal Ca2+ release by caffeine is due to the regulation of Ca2+ release by the lumenal [Ca2+], and suggests that changes in [Ca2+]c do not play a major role in the development of the quantal effect.


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)

Regulation of caffeine-induced Ca2+ release by  [Ca2+]ER. The ER was refilled by perfusing with medium containing 1 mM Ca2+ and then 1 μM histamine or different concentrations of caffeine were added, as indicated. In the experiment  shown in c, cells were incubated with 10 μM BAPTA-AM during  aequorin reconstitution in order to load the cytosol with this  Ca2+ chelator. Other details are as in Fig. 1.
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Related In: Results  -  Collection

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Figure 4: Regulation of caffeine-induced Ca2+ release by [Ca2+]ER. The ER was refilled by perfusing with medium containing 1 mM Ca2+ and then 1 μM histamine or different concentrations of caffeine were added, as indicated. In the experiment shown in c, cells were incubated with 10 μM BAPTA-AM during aequorin reconstitution in order to load the cytosol with this Ca2+ chelator. Other details are as in Fig. 1.
Mentions: The experiments of Fig. 3 are consistent with previous results obtained looking at [Ca2+]c in the same cell preparation by Cheek et al., 1994a. Those experiments led the authors to suggest that there should be different compartments within the ER having different sensitivities to caffeine. This hypothesis would explain why ryanodine only empties the pool sensitive to 5 mM caffeine but leaves the rest of the pool untouched, which could be only released with a higher caffeine dose. However, there is an alternative explanation for these results, based on the regulation of caffeine-induced Ca2+ release by the lumenal [Ca2+]. In this hypothesis, the results can be explained with only one ER compartment if we assume that submaximal caffeine concentrations can only release Ca2+ until [Ca2+]ER is reduced to a certain level. The higher the caffeine concentration, the lower the [Ca2+]ER level attained. Both alternative hypotheses lead to different predictions under some experimental conditions. In particular, if we obtain half-filled stores by different procedures, e.g., by emptying them with an agonist acting via InsP3 production or by refilling the stores only halfway, the hypothesis of several compartments predicts that we should have all of them half-filled. Therefore, a submaximal dose of caffeine should still release Ca2+ from half of them. On the contrary, if the release was directly controlled by [Ca2+]ER, we would expect the effect of 5 mM caffeine to be independent of the procedure used to reach that half-filling. The experiments shown in Fig. 4 indicate that the last hypothesis is the correct one. In Fig. 4 a, half-filling was obtained by emptying the ER with histamine. After that, 5 mM caffeine had no effect. Instead, if 5 mM caffeine was added with the ER full of Ca2+, it was able to empty it exactly down to the same point. Fig. 4 b shows the effect of half-filling the ER by reducing the time of refilling. Again, the effect of 5 mM caffeine was strictly dependent on the level of [Ca2+]ER reached at the point it was added. It had no effect at half-filling, but released 50% of the pool when the Ca2+ stores were completely filled. Fig. 4 c shows a similar approach but made in cells preloaded with the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) to slow the refilling. We can observe again that the effect of 5 mM caffeine was strictly dependent on the [Ca2+]ER at the moment of addition. In addition, this experiment also shows that Ca2+ release induced by caffeine requires only resting [Ca2+]c. Fura-2 measurements performed in parallel showed that in cells loaded with BAPTA, the [Ca2+]c changes induced by caffeine were almost abolished (data not shown). This result points out also that quantal Ca2+ release by caffeine is due to the regulation of Ca2+ release by the lumenal [Ca2+], and suggests that changes in [Ca2+]c do not play a major role in the development of the quantal effect.

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