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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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Quantal response to submaximal concentrations of caffeine. The ER  was refilled by perfusing with medium  containing 1 mM Ca2+ either in the presence (b) or in the absence of caffeine.  Then, different concentrations of caffeine  were added as indicated, either in the  presence or in the absence of 10 μM ryanodine. Other details are as in Fig. 1.
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Figure 3: Quantal response to submaximal concentrations of caffeine. The ER was refilled by perfusing with medium containing 1 mM Ca2+ either in the presence (b) or in the absence of caffeine. Then, different concentrations of caffeine were added as indicated, either in the presence or in the absence of 10 μM ryanodine. Other details are as in Fig. 1.

Mentions: The effect of caffeine has been reported to be quantal (Cheek et al., 1993, 1994a), meaning that low caffeine concentrations release only part of the caffeine-sensitive pool. The same phenomenon was observed here. Fig. 3 a shows that addition of submaximal caffeine concentrations induced a rapid but partial emptying of the ER, leading within 30 s to a new lower steady-state of [Ca2+]ER. At that point, only the addition of a higher caffeine concentration was able to produce further emptying of the ER. Similar quantal effects were also observed during refilling of the ER when it was carried out in the presence of caffeine. Fig. 3 b shows that the ER did not refill in the presence of 50 mM caffeine, but refilled about halfway once the caffeine concentration was dropped to 5 mM, and completely when caffeine was washed away. Subsequent addition of 5 and 50 mM caffeine released Ca2+ and reached the same [Ca2+]ER levels obtained during refilling in the presence of these caffeine concentrations. The degree of emptying induced by a particular caffeine concentration was quite reproducible in consecutive additions. Fig 3 c shows that consecutive additions of 5 mM caffeine produced always ∼50% emptying of the ER, and only the addition of a higher caffeine concentration was able to produce further emptying. Fig. 3 d shows the effect of ryanodine added in the presence of a submaximal dose of caffeine. We can see that the first pulses were identical to the control, but again here the ER refilled progressively more slowly after each new caffeine addition. In this case, in contrast to the experiment shown in Fig. 2, finally a [Ca2+]ER steady-state corresponding to about half-filling was reached, in fact, the same [Ca2+]ER obtained initially after addition of 5 mM caffeine. Once at this point, addition of a maximal dose of caffeine was required to induce emptying of the remaining portion of the 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)

Quantal response to submaximal concentrations of caffeine. The ER  was refilled by perfusing with medium  containing 1 mM Ca2+ either in the presence (b) or in the absence of caffeine.  Then, different concentrations of caffeine  were added as indicated, either in the  presence or in the absence of 10 μM ryanodine. Other details are as in Fig. 1.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Quantal response to submaximal concentrations of caffeine. The ER was refilled by perfusing with medium containing 1 mM Ca2+ either in the presence (b) or in the absence of caffeine. Then, different concentrations of caffeine were added as indicated, either in the presence or in the absence of 10 μM ryanodine. Other details are as in Fig. 1.
Mentions: The effect of caffeine has been reported to be quantal (Cheek et al., 1993, 1994a), meaning that low caffeine concentrations release only part of the caffeine-sensitive pool. The same phenomenon was observed here. Fig. 3 a shows that addition of submaximal caffeine concentrations induced a rapid but partial emptying of the ER, leading within 30 s to a new lower steady-state of [Ca2+]ER. At that point, only the addition of a higher caffeine concentration was able to produce further emptying of the ER. Similar quantal effects were also observed during refilling of the ER when it was carried out in the presence of caffeine. Fig. 3 b shows that the ER did not refill in the presence of 50 mM caffeine, but refilled about halfway once the caffeine concentration was dropped to 5 mM, and completely when caffeine was washed away. Subsequent addition of 5 and 50 mM caffeine released Ca2+ and reached the same [Ca2+]ER levels obtained during refilling in the presence of these caffeine concentrations. The degree of emptying induced by a particular caffeine concentration was quite reproducible in consecutive additions. Fig 3 c shows that consecutive additions of 5 mM caffeine produced always ∼50% emptying of the ER, and only the addition of a higher caffeine concentration was able to produce further emptying. Fig. 3 d shows the effect of ryanodine added in the presence of a submaximal dose of caffeine. We can see that the first pulses were identical to the control, but again here the ER refilled progressively more slowly after each new caffeine addition. In this case, in contrast to the experiment shown in Fig. 2, finally a [Ca2+]ER steady-state corresponding to about half-filling was reached, in fact, the same [Ca2+]ER obtained initially after addition of 5 mM caffeine. Once at this point, addition of a maximal dose of caffeine was required to induce emptying of the remaining portion of the 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