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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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Use-dependent inhibition of ER refilling by ryanodine.  The ER was refilled by perfusing with medium containing 1 mM  Ca2+. Then, several consecutive stimulations with 50 mM caffeine were performed as indicated, either in the absence (a) or in  the presence (b) of 10 μM ryanodine. In b, standard medium containing 70 mM KCl (replacing an equimolar amount of NaCl)  was perfused when indicated (K+). Other details are as in Fig. 1.
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Figure 2: Use-dependent inhibition of ER refilling by ryanodine. The ER was refilled by perfusing with medium containing 1 mM Ca2+. Then, several consecutive stimulations with 50 mM caffeine were performed as indicated, either in the absence (a) or in the presence (b) of 10 μM ryanodine. In b, standard medium containing 70 mM KCl (replacing an equimolar amount of NaCl) was perfused when indicated (K+). Other details are as in Fig. 1.

Mentions: Fig. 2 illustrates the time course of the use-dependent effect of ryanodine on RyR. Fig. 2 a shows that consecutive additions of 50 mM caffeine produced comparable decreases in [Ca2+]ER if an interval of 3–5 min was left between two consecutive additions to allow refilling with Ca2+ of the ER. If 10 μM ryanodine was present during the caffeine pulses (Fig. 2 b), the first pulse was identical to the control but then the ER became progressively unable to refill. After four pulses, the ER remained at near background [Ca2+]ER levels, and only the increase in [Ca2+]c elicited by depolarization with 70 mM K+ was able to activate the Ca2+ pump and produce a small and transient increase in [Ca2+]ER. The effect of caffeine was not inhibited by incubation with 20 μM dantrolene (data not shown), an inhibitor of RyR that is particularly effective on the skeletal muscle RyR1 (Van Winkle, 1976).


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)

Use-dependent inhibition of ER refilling by ryanodine.  The ER was refilled by perfusing with medium containing 1 mM  Ca2+. Then, several consecutive stimulations with 50 mM caffeine were performed as indicated, either in the absence (a) or in  the presence (b) of 10 μM ryanodine. In b, standard medium containing 70 mM KCl (replacing an equimolar amount of NaCl)  was perfused when indicated (K+). Other details are as in Fig. 1.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Use-dependent inhibition of ER refilling by ryanodine. The ER was refilled by perfusing with medium containing 1 mM Ca2+. Then, several consecutive stimulations with 50 mM caffeine were performed as indicated, either in the absence (a) or in the presence (b) of 10 μM ryanodine. In b, standard medium containing 70 mM KCl (replacing an equimolar amount of NaCl) was perfused when indicated (K+). Other details are as in Fig. 1.
Mentions: Fig. 2 illustrates the time course of the use-dependent effect of ryanodine on RyR. Fig. 2 a shows that consecutive additions of 50 mM caffeine produced comparable decreases in [Ca2+]ER if an interval of 3–5 min was left between two consecutive additions to allow refilling with Ca2+ of the ER. If 10 μM ryanodine was present during the caffeine pulses (Fig. 2 b), the first pulse was identical to the control but then the ER became progressively unable to refill. After four pulses, the ER remained at near background [Ca2+]ER levels, and only the increase in [Ca2+]c elicited by depolarization with 70 mM K+ was able to activate the Ca2+ pump and produce a small and transient increase in [Ca2+]ER. The effect of caffeine was not inhibited by incubation with 20 μM dantrolene (data not shown), an inhibitor of RyR that is particularly effective on the skeletal muscle RyR1 (Van Winkle, 1976).

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