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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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(a) Effects of cADPR, InsP3, and caffeine on [Ca2+]ER  in permeabilized cells. Cells were permeabilized by perfusion  with 20 μM digitonin for 1 min as indicated. Then the ER was refilled by perfusion with medium containing 100 nM Ca2+ (buffered with EGTA). Finally, either 5 μM cADPR, 2 μM InsP3, or  50 mM caffeine were perfused as indicated. (b) Effect of preincubation with 20 nM thapsigargin on the [Ca2+]c responses to histamine, caffeine, and high K+ medium. Cells were loaded with  fura-2, preincubated for 20 min with 20 nM thapsigargin, and  then suspended in standard medium containing 1 mM CaCl2.  Then, either 10 μM histamine, 50 mM caffeine, or standard medium containing 1 mM CaCl2 and 70 mM KCl were perfused as  indicated. Both histamine and caffeine were perfused in Ca2+-free medium (containing 100 μM EGTA) to avoid Ca2+ entry.  Perfusion with Ca2+-free medium was started 15 s before and  continued for 15 s after stimulation with histamine or caffeine.  The trace shown corresponds to the average of 42 cells present in  the microscope field. Other details are as in Fig. 1.
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Figure 6: (a) Effects of cADPR, InsP3, and caffeine on [Ca2+]ER in permeabilized cells. Cells were permeabilized by perfusion with 20 μM digitonin for 1 min as indicated. Then the ER was refilled by perfusion with medium containing 100 nM Ca2+ (buffered with EGTA). Finally, either 5 μM cADPR, 2 μM InsP3, or 50 mM caffeine were perfused as indicated. (b) Effect of preincubation with 20 nM thapsigargin on the [Ca2+]c responses to histamine, caffeine, and high K+ medium. Cells were loaded with fura-2, preincubated for 20 min with 20 nM thapsigargin, and then suspended in standard medium containing 1 mM CaCl2. Then, either 10 μM histamine, 50 mM caffeine, or standard medium containing 1 mM CaCl2 and 70 mM KCl were perfused as indicated. Both histamine and caffeine were perfused in Ca2+-free medium (containing 100 μM EGTA) to avoid Ca2+ entry. Perfusion with Ca2+-free medium was started 15 s before and continued for 15 s after stimulation with histamine or caffeine. The trace shown corresponds to the average of 42 cells present in the microscope field. Other details are as in Fig. 1.

Mentions: The effect of a low caffeine concentration as positive modulator of CICR might reproduce the action of a physiological modulator of this phenomenon. Phosphorylation by cAMP-dependent protein kinase and production of the β-NAD+ metabolite cyclic ADP ribose (cADPR) have been reported to act as physiological modulators for RyR in bovine chromaffin cells (Morita et al., 1997). However, incubation for 3–5 min with the adenylate cyclase activator forskolin (20 μM) had neither any significant effect on the sensitivity to caffeine of Ca2+ release nor on the magnitude of high K+ depolarization-induced CICR (data not shown). Regarding cADPR, it has been reported that acetylcholine, high K+ depolarization, and forskolin all stimulate its synthesis by ADP ribosyl cyclase in bovine chromaffin cells (Morita et al., 1997). Therefore, production of this mediator should already be stimulated in our CICR experiments. Nevertheless, to study directly the effect of cADPR on Ca2+ release from the ER, we performed experiments in permeabilized cells. Cells were depleted of Ca2+ and reconstituted with coelenterazine as usual. Recording of luminescence was started and the cells were permeabilized by perfusion with intracellular-like medium containing 20 μM digitonin and 2 mM EGTA for 1 min. Then, intracellular-like medium containing 100 nM Ca2+ (buffered with EGTA) and 2 mM ATP-Mg was perfused. Fig. 6 a shows that [Ca2+]ER increased in digitonin-permeabilized cells with very similar kinetics to that found in intact cells after addition of 1 mM extracellular Ca2+. The steady-state [Ca2+]ER reached was also similar (compare with Fig. 1 a). Fig. 6 a also shows that 2 μM InsP3 and 50 mM caffeine produced a rapid and near complete release of Ca2+ from the ER whereas 5 μM cADPR had no effect. Two different commercial sources of cADPR were tested with the same results. In some experiments, cADPR was added in the presence of 1 μM calmodulin and no effect was found either. A possible explanation for the discrepancy among our results and those of Morita et al. (1997) would be that cADPR releases Ca2+ from a different (non-ER) Ca2+ pool. In fact, these authors report that InsP3 releases Ca2+ from a pool sensitive to 20 nM thapsigargin, whereas cADPR and caffeine release Ca2+ from a pool only sensitive to >200 nM thapsigargin. In our hands, refilling of the ER was completely inhibited by either 20 nM or 1 μM thapsigargin in both intact (see above) and permeabilized cells (data not shown). Therefore, in order to make compatible our own results and those of Morita et al. (1997), cADPR and caffeine should be able to release Ca2+ from an additional non-ER Ca2+ pool in the presence of 20 nM thapsigargin. We then performed single-cell fura-2 imaging experiments in intact cells looking at the effect of caffeine on [Ca2+]c in cells pretreated with 20 nM thapsigargin. Fig. 6 b shows that addition of caffeine or histamine in Ca2+-free medium produced no increase in [Ca2+]c under these conditions (the observed decrease is due to the perfusion of Ca2+-free medium), whereas K+ depolarization still produced the usual [Ca2+]c peak due to Ca2+ entry.


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)

(a) Effects of cADPR, InsP3, and caffeine on [Ca2+]ER  in permeabilized cells. Cells were permeabilized by perfusion  with 20 μM digitonin for 1 min as indicated. Then the ER was refilled by perfusion with medium containing 100 nM Ca2+ (buffered with EGTA). Finally, either 5 μM cADPR, 2 μM InsP3, or  50 mM caffeine were perfused as indicated. (b) Effect of preincubation with 20 nM thapsigargin on the [Ca2+]c responses to histamine, caffeine, and high K+ medium. Cells were loaded with  fura-2, preincubated for 20 min with 20 nM thapsigargin, and  then suspended in standard medium containing 1 mM CaCl2.  Then, either 10 μM histamine, 50 mM caffeine, or standard medium containing 1 mM CaCl2 and 70 mM KCl were perfused as  indicated. Both histamine and caffeine were perfused in Ca2+-free medium (containing 100 μM EGTA) to avoid Ca2+ entry.  Perfusion with Ca2+-free medium was started 15 s before and  continued for 15 s after stimulation with histamine or caffeine.  The trace shown corresponds to the average of 42 cells present in  the microscope field. Other details are as in Fig. 1.
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Figure 6: (a) Effects of cADPR, InsP3, and caffeine on [Ca2+]ER in permeabilized cells. Cells were permeabilized by perfusion with 20 μM digitonin for 1 min as indicated. Then the ER was refilled by perfusion with medium containing 100 nM Ca2+ (buffered with EGTA). Finally, either 5 μM cADPR, 2 μM InsP3, or 50 mM caffeine were perfused as indicated. (b) Effect of preincubation with 20 nM thapsigargin on the [Ca2+]c responses to histamine, caffeine, and high K+ medium. Cells were loaded with fura-2, preincubated for 20 min with 20 nM thapsigargin, and then suspended in standard medium containing 1 mM CaCl2. Then, either 10 μM histamine, 50 mM caffeine, or standard medium containing 1 mM CaCl2 and 70 mM KCl were perfused as indicated. Both histamine and caffeine were perfused in Ca2+-free medium (containing 100 μM EGTA) to avoid Ca2+ entry. Perfusion with Ca2+-free medium was started 15 s before and continued for 15 s after stimulation with histamine or caffeine. The trace shown corresponds to the average of 42 cells present in the microscope field. Other details are as in Fig. 1.
Mentions: The effect of a low caffeine concentration as positive modulator of CICR might reproduce the action of a physiological modulator of this phenomenon. Phosphorylation by cAMP-dependent protein kinase and production of the β-NAD+ metabolite cyclic ADP ribose (cADPR) have been reported to act as physiological modulators for RyR in bovine chromaffin cells (Morita et al., 1997). However, incubation for 3–5 min with the adenylate cyclase activator forskolin (20 μM) had neither any significant effect on the sensitivity to caffeine of Ca2+ release nor on the magnitude of high K+ depolarization-induced CICR (data not shown). Regarding cADPR, it has been reported that acetylcholine, high K+ depolarization, and forskolin all stimulate its synthesis by ADP ribosyl cyclase in bovine chromaffin cells (Morita et al., 1997). Therefore, production of this mediator should already be stimulated in our CICR experiments. Nevertheless, to study directly the effect of cADPR on Ca2+ release from the ER, we performed experiments in permeabilized cells. Cells were depleted of Ca2+ and reconstituted with coelenterazine as usual. Recording of luminescence was started and the cells were permeabilized by perfusion with intracellular-like medium containing 20 μM digitonin and 2 mM EGTA for 1 min. Then, intracellular-like medium containing 100 nM Ca2+ (buffered with EGTA) and 2 mM ATP-Mg was perfused. Fig. 6 a shows that [Ca2+]ER increased in digitonin-permeabilized cells with very similar kinetics to that found in intact cells after addition of 1 mM extracellular Ca2+. The steady-state [Ca2+]ER reached was also similar (compare with Fig. 1 a). Fig. 6 a also shows that 2 μM InsP3 and 50 mM caffeine produced a rapid and near complete release of Ca2+ from the ER whereas 5 μM cADPR had no effect. Two different commercial sources of cADPR were tested with the same results. In some experiments, cADPR was added in the presence of 1 μM calmodulin and no effect was found either. A possible explanation for the discrepancy among our results and those of Morita et al. (1997) would be that cADPR releases Ca2+ from a different (non-ER) Ca2+ pool. In fact, these authors report that InsP3 releases Ca2+ from a pool sensitive to 20 nM thapsigargin, whereas cADPR and caffeine release Ca2+ from a pool only sensitive to >200 nM thapsigargin. In our hands, refilling of the ER was completely inhibited by either 20 nM or 1 μM thapsigargin in both intact (see above) and permeabilized cells (data not shown). Therefore, in order to make compatible our own results and those of Morita et al. (1997), cADPR and caffeine should be able to release Ca2+ from an additional non-ER Ca2+ pool in the presence of 20 nM thapsigargin. We then performed single-cell fura-2 imaging experiments in intact cells looking at the effect of caffeine on [Ca2+]c in cells pretreated with 20 nM thapsigargin. Fig. 6 b shows that addition of caffeine or histamine in Ca2+-free medium produced no increase in [Ca2+]c under these conditions (the observed decrease is due to the perfusion of Ca2+-free medium), whereas K+ depolarization still produced the usual [Ca2+]c peak due to Ca2+ entry.

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