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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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Effects of histamine and caffeine and pretreatment  with ryanodine or thapsigargin on [Ca2+]ER. HSV-1–infected  chromaffin cells were depleted of Ca2+ and reconstituted with  coelenterazine n. (a) The ER was refilled by incubation with medium containing 1 mM Ca2+, then either 10 μM histamine or  50 mM caffeine were perfused as indicated. (b) Where indicated,  ryanodine-pretreated cells were treated before aequorin reconstitution with medium containing 50 mM caffeine and 10 μM  ryanodine for 2 min. Cells were then washed and the same treatment was repeated four times at 2-min intervals. Where indicated,  thapsigargin-pretreated cells were incubated with 1 μM thapsigargin for 10 min before starting the record. During the experiments,  medium containing 1 mM Ca2+ and either 50 mM caffeine, 1 μM  bradykinin, or 10 μM histamine was perfused as indicated.
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Figure 1: Effects of histamine and caffeine and pretreatment with ryanodine or thapsigargin on [Ca2+]ER. HSV-1–infected chromaffin cells were depleted of Ca2+ and reconstituted with coelenterazine n. (a) The ER was refilled by incubation with medium containing 1 mM Ca2+, then either 10 μM histamine or 50 mM caffeine were perfused as indicated. (b) Where indicated, ryanodine-pretreated cells were treated before aequorin reconstitution with medium containing 50 mM caffeine and 10 μM ryanodine for 2 min. Cells were then washed and the same treatment was repeated four times at 2-min intervals. Where indicated, thapsigargin-pretreated cells were incubated with 1 μM thapsigargin for 10 min before starting the record. During the experiments, medium containing 1 mM Ca2+ and either 50 mM caffeine, 1 μM bradykinin, or 10 μM histamine was perfused as indicated.

Mentions: After aequorin reconstitution with coelenterazine, with the ER completely depleted of Ca2+, the experiments were started by perfusing the cells with medium containing 1 mM Ca2+ to refill the ER (Fig. 1 a). As in other cells studied previously (Montero et al., 1995, 1997a; Barrero et al., 1997; Alonso et al., 1998), full refilling of the ER required 3–5 min and the steady-state [Ca2+]ER reached was 500–800 μM (Fig. 1 a). Addition of histamine produced a rapid but partial (60–80%) Ca2+ emptying of the ER, a new [Ca2+]ER steady-state being reached at ∼200–300 μM. Subsequent addition of caffeine (50 mM) induced a further emptying to near background aequorin luminescence. The effects were reversible by washing, this allowing refilling of the ER that was completed within 3–5 min. Addition of caffeine at that point, when the stores were completely refilled, triggered a rapid and complete emptying of the ER. Histamine was then unable to produce any further effect. These results suggest that essentially the whole ER Ca2+ pool is sensitive to caffeine and a large part of it is also sensitive to InsP3 producing agonists such as histamine. Similar results were obtained using 1 μM bradykinin instead of histamine (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)

Effects of histamine and caffeine and pretreatment  with ryanodine or thapsigargin on [Ca2+]ER. HSV-1–infected  chromaffin cells were depleted of Ca2+ and reconstituted with  coelenterazine n. (a) The ER was refilled by incubation with medium containing 1 mM Ca2+, then either 10 μM histamine or  50 mM caffeine were perfused as indicated. (b) Where indicated,  ryanodine-pretreated cells were treated before aequorin reconstitution with medium containing 50 mM caffeine and 10 μM  ryanodine for 2 min. Cells were then washed and the same treatment was repeated four times at 2-min intervals. Where indicated,  thapsigargin-pretreated cells were incubated with 1 μM thapsigargin for 10 min before starting the record. During the experiments,  medium containing 1 mM Ca2+ and either 50 mM caffeine, 1 μM  bradykinin, or 10 μM histamine was perfused as indicated.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132888&req=5

Figure 1: Effects of histamine and caffeine and pretreatment with ryanodine or thapsigargin on [Ca2+]ER. HSV-1–infected chromaffin cells were depleted of Ca2+ and reconstituted with coelenterazine n. (a) The ER was refilled by incubation with medium containing 1 mM Ca2+, then either 10 μM histamine or 50 mM caffeine were perfused as indicated. (b) Where indicated, ryanodine-pretreated cells were treated before aequorin reconstitution with medium containing 50 mM caffeine and 10 μM ryanodine for 2 min. Cells were then washed and the same treatment was repeated four times at 2-min intervals. Where indicated, thapsigargin-pretreated cells were incubated with 1 μM thapsigargin for 10 min before starting the record. During the experiments, medium containing 1 mM Ca2+ and either 50 mM caffeine, 1 μM bradykinin, or 10 μM histamine was perfused as indicated.
Mentions: After aequorin reconstitution with coelenterazine, with the ER completely depleted of Ca2+, the experiments were started by perfusing the cells with medium containing 1 mM Ca2+ to refill the ER (Fig. 1 a). As in other cells studied previously (Montero et al., 1995, 1997a; Barrero et al., 1997; Alonso et al., 1998), full refilling of the ER required 3–5 min and the steady-state [Ca2+]ER reached was 500–800 μM (Fig. 1 a). Addition of histamine produced a rapid but partial (60–80%) Ca2+ emptying of the ER, a new [Ca2+]ER steady-state being reached at ∼200–300 μM. Subsequent addition of caffeine (50 mM) induced a further emptying to near background aequorin luminescence. The effects were reversible by washing, this allowing refilling of the ER that was completed within 3–5 min. Addition of caffeine at that point, when the stores were completely refilled, triggered a rapid and complete emptying of the ER. Histamine was then unable to produce any further effect. These results suggest that essentially the whole ER Ca2+ pool is sensitive to caffeine and a large part of it is also sensitive to InsP3 producing agonists such as histamine. Similar results were obtained using 1 μM bradykinin instead of histamine (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