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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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Effect of [Ca2+]ER on CICR induced by depolarization  with high K+. Cells depleted of Ca2+ and reconstituted with coelenterazine n were placed in the luminometer in 0.5 mM EGTA  containing standard medium. Then, 10-s pulses of medium containing 70 mM KCl and 2 mM CaCl2 were given as indicated and  0.5 mM EGTA containing standard medium was perfused during  the intervals. After five pulses, Ca2+-containing (1 mM) medium  was perfused for 3 min to refill the ER, and then the previous  protocol was started again.
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Figure 8: Effect of [Ca2+]ER on CICR induced by depolarization with high K+. Cells depleted of Ca2+ and reconstituted with coelenterazine n were placed in the luminometer in 0.5 mM EGTA containing standard medium. Then, 10-s pulses of medium containing 70 mM KCl and 2 mM CaCl2 were given as indicated and 0.5 mM EGTA containing standard medium was perfused during the intervals. After five pulses, Ca2+-containing (1 mM) medium was perfused for 3 min to refill the ER, and then the previous protocol was started again.

Mentions: In spite of the lack of inhibition by ryanodine, the potentiation by caffeine of Ca2+ release induced by K+ depolarization (as shown in Fig. 5 d) suggests that CICR may take place through the same RyR activated by caffeine. An important property of the RyR activated by caffeine is the regulation by lumenal [Ca2+] shown in Figs. 3 and 4. In the case of CICR, data of Fig. 5 b also suggest that Ca2+ release may be stronger at higher lumenal [Ca2+] since the first stimulation with high K+, carried out when the ER was only half-filled, was less efficient than the subsequent ones. To obtain more evidence on this point, we investigated the effects of 10-s pulses of high K+/Ca2+-containing medium at different [Ca2+]ER levels (Fig. 8). At the beginning of the experiment the ER was completely depleted of Ca2+ and the first two K+ pulses induced an increase in [Ca2+]ER. Since the cells were kept in EGTA-containing medium during the intervals between the K+/Ca2+ pulses, refilling of the ER took place only from Ca2+ entering into the cells during the pulses. Subsequent K+ pulses produced also [Ca2+]ER increase, although progressively smaller, reaching finally a steady-state [Ca2+]ER at ∼300 μM, where the K+/Ca2+ pulses had almost no effect. We then completely refilled the ER with Ca2+ by perfusing the cells with Ca2+-containing medium, thus reaching the usual [Ca2+]ER steady-state levels at ∼700 μM. At that point, we restarted the protocol of repeated stimulation with 10-s high K+/Ca2+ pulses, using EGTA-containing medium for the intervals. The pulses produced now a rapid Ca2+ release that could be clearly distinguished from the slower [Ca2+]ER decrease induced by the lack of extracellular Ca2+. After three K+ pulses, [Ca2+]ER was reduced again to ∼300 μM, and at that point the last pulse produced little effect. This result shows clearly that Ca2+ release induced by K+ depolarization, similarly to that induced by caffeine, is strictly dependent on the [Ca2+]ER.


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)

Effect of [Ca2+]ER on CICR induced by depolarization  with high K+. Cells depleted of Ca2+ and reconstituted with coelenterazine n were placed in the luminometer in 0.5 mM EGTA  containing standard medium. Then, 10-s pulses of medium containing 70 mM KCl and 2 mM CaCl2 were given as indicated and  0.5 mM EGTA containing standard medium was perfused during  the intervals. After five pulses, Ca2+-containing (1 mM) medium  was perfused for 3 min to refill the ER, and then the previous  protocol was started again.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132888&req=5

Figure 8: Effect of [Ca2+]ER on CICR induced by depolarization with high K+. Cells depleted of Ca2+ and reconstituted with coelenterazine n were placed in the luminometer in 0.5 mM EGTA containing standard medium. Then, 10-s pulses of medium containing 70 mM KCl and 2 mM CaCl2 were given as indicated and 0.5 mM EGTA containing standard medium was perfused during the intervals. After five pulses, Ca2+-containing (1 mM) medium was perfused for 3 min to refill the ER, and then the previous protocol was started again.
Mentions: In spite of the lack of inhibition by ryanodine, the potentiation by caffeine of Ca2+ release induced by K+ depolarization (as shown in Fig. 5 d) suggests that CICR may take place through the same RyR activated by caffeine. An important property of the RyR activated by caffeine is the regulation by lumenal [Ca2+] shown in Figs. 3 and 4. In the case of CICR, data of Fig. 5 b also suggest that Ca2+ release may be stronger at higher lumenal [Ca2+] since the first stimulation with high K+, carried out when the ER was only half-filled, was less efficient than the subsequent ones. To obtain more evidence on this point, we investigated the effects of 10-s pulses of high K+/Ca2+-containing medium at different [Ca2+]ER levels (Fig. 8). At the beginning of the experiment the ER was completely depleted of Ca2+ and the first two K+ pulses induced an increase in [Ca2+]ER. Since the cells were kept in EGTA-containing medium during the intervals between the K+/Ca2+ pulses, refilling of the ER took place only from Ca2+ entering into the cells during the pulses. Subsequent K+ pulses produced also [Ca2+]ER increase, although progressively smaller, reaching finally a steady-state [Ca2+]ER at ∼300 μM, where the K+/Ca2+ pulses had almost no effect. We then completely refilled the ER with Ca2+ by perfusing the cells with Ca2+-containing medium, thus reaching the usual [Ca2+]ER steady-state levels at ∼700 μM. At that point, we restarted the protocol of repeated stimulation with 10-s high K+/Ca2+ pulses, using EGTA-containing medium for the intervals. The pulses produced now a rapid Ca2+ release that could be clearly distinguished from the slower [Ca2+]ER decrease induced by the lack of extracellular Ca2+. After three K+ pulses, [Ca2+]ER was reduced again to ∼300 μM, and at that point the last pulse produced little effect. This result shows clearly that Ca2+ release induced by K+ depolarization, similarly to that induced by caffeine, is strictly dependent on the [Ca2+]ER.

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