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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 ryanodine and either depolarization with high  K+ medium (a), field electric stimulation (b), or histamine (c) on  the [Ca2+]c responses induced by caffeine. Cells were loaded with  fura-2 and placed under the microscope in standard medium containing 1 mM CaCl2. Then, different stimuli were given as indicated: different concentrations of caffeine (Caf, in mM), medium  containing 70 mM KCl (K+), 10 μM histamine (His), 10 μM ryanodine (Ry), or field electric stimulation (E.S. 10 Hz for 10 s, arrows). Caffeine and histamine were added in Ca2+-free medium  (containing 100 μM EGTA). Perfusion with Ca2+ free medium  was started 15 s before and continued for 15 s after the stimuli.  Transition from Ca2+-containing to Ca2+-free medium sometimes  produced a small [Ca2+]c peak. The traces shown are the average  of 36 (a), 49 (b), and 61 (c) cells present in the microscope field.
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Figure 7: Effects of ryanodine and either depolarization with high K+ medium (a), field electric stimulation (b), or histamine (c) on the [Ca2+]c responses induced by caffeine. Cells were loaded with fura-2 and placed under the microscope in standard medium containing 1 mM CaCl2. Then, different stimuli were given as indicated: different concentrations of caffeine (Caf, in mM), medium containing 70 mM KCl (K+), 10 μM histamine (His), 10 μM ryanodine (Ry), or field electric stimulation (E.S. 10 Hz for 10 s, arrows). Caffeine and histamine were added in Ca2+-free medium (containing 100 μM EGTA). Perfusion with Ca2+ free medium was started 15 s before and continued for 15 s after the stimuli. Transition from Ca2+-containing to Ca2+-free medium sometimes produced a small [Ca2+]c peak. The traces shown are the average of 36 (a), 49 (b), and 61 (c) cells present in the microscope field.

Mentions: On the basis of the use-sensitive action of ryanodine, illustrated here for caffeine stimulation (Figs. 2 and 3), we should expect also to find a use-sensitive inhibition of ER refilling after repeated stimulation with high K+ pulses inducing CICR. Under this rationale, experiments similar to those shown in Fig. 5, but in the presence of 10 μM ryanodine, were performed. In these experiments we were not able to detect any significant effect of ryanodine on the [Ca2+]ER decrease induced by four or five consecutive K+ pulses (data not shown). However, as aequorin consumption limits the sensitivity of the measurement at the end of these experiments, we also decided to perform single-cell imaging experiments looking at the magnitude of the [Ca2+]c peak induced by caffeine after several K+ pulses in the presence of ryanodine. Fig 7 a shows that the [Ca2+]c increase induced by caffeine was little modified after five consecutive 30-s K+ pulses given in the presence of ryanodine (compare with the initial three caffeine additions). It is interesting to note that the second caffeine addition after the K+ + ryanodine pulses produced no [Ca2+]c increase, even though caffeine was always added in the absence of ryanodine. In fact, perfusion with ryanodine for a short period (30–60 s) before any caffeine addition was effective in promoting the typical use-dependent inhibition of ER refilling on application of caffeine (without ryanodine) 30–60 min later. This means that, even though ryanodine by itself does not produce any apparent effect on ER filling, it remains within the cells after washing and acts later, on stimulation of Ca2+ release (see below). Depolarization can also be produced using the nicotinic acetylcholine agonist DMPP instead of K+. Again here, consecutive pulses of 10 μM DMPP in the presence of ryanodine did not have any effect on a later [Ca2+]c peak induced by caffeine (data not shown). An alternative and perhaps more physiological depolarizing maneuver is field electric stimulation. Fig. 7 b shows the effect of several consecutive 10-s pulses at 10 Hz, before and after the addition of ryanodine. Again, we can observe that 5 mM caffeine produced the same [Ca2+]c peak after and before field electric stimulation. As in Fig. 7 a, consecutive additions of caffeine produced inhibition long after washing of ryanodine. The lack of effect of ryanodine in these experiments may be attributed to the much smaller activation of Ca2+ release by high K+-induced Ca2+ entry compared with that induced by caffeine.


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 ryanodine and either depolarization with high  K+ medium (a), field electric stimulation (b), or histamine (c) on  the [Ca2+]c responses induced by caffeine. Cells were loaded with  fura-2 and placed under the microscope in standard medium containing 1 mM CaCl2. Then, different stimuli were given as indicated: different concentrations of caffeine (Caf, in mM), medium  containing 70 mM KCl (K+), 10 μM histamine (His), 10 μM ryanodine (Ry), or field electric stimulation (E.S. 10 Hz for 10 s, arrows). Caffeine and histamine were added in Ca2+-free medium  (containing 100 μM EGTA). Perfusion with Ca2+ free medium  was started 15 s before and continued for 15 s after the stimuli.  Transition from Ca2+-containing to Ca2+-free medium sometimes  produced a small [Ca2+]c peak. The traces shown are the average  of 36 (a), 49 (b), and 61 (c) cells present in the microscope field.
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Related In: Results  -  Collection

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Figure 7: Effects of ryanodine and either depolarization with high K+ medium (a), field electric stimulation (b), or histamine (c) on the [Ca2+]c responses induced by caffeine. Cells were loaded with fura-2 and placed under the microscope in standard medium containing 1 mM CaCl2. Then, different stimuli were given as indicated: different concentrations of caffeine (Caf, in mM), medium containing 70 mM KCl (K+), 10 μM histamine (His), 10 μM ryanodine (Ry), or field electric stimulation (E.S. 10 Hz for 10 s, arrows). Caffeine and histamine were added in Ca2+-free medium (containing 100 μM EGTA). Perfusion with Ca2+ free medium was started 15 s before and continued for 15 s after the stimuli. Transition from Ca2+-containing to Ca2+-free medium sometimes produced a small [Ca2+]c peak. The traces shown are the average of 36 (a), 49 (b), and 61 (c) cells present in the microscope field.
Mentions: On the basis of the use-sensitive action of ryanodine, illustrated here for caffeine stimulation (Figs. 2 and 3), we should expect also to find a use-sensitive inhibition of ER refilling after repeated stimulation with high K+ pulses inducing CICR. Under this rationale, experiments similar to those shown in Fig. 5, but in the presence of 10 μM ryanodine, were performed. In these experiments we were not able to detect any significant effect of ryanodine on the [Ca2+]ER decrease induced by four or five consecutive K+ pulses (data not shown). However, as aequorin consumption limits the sensitivity of the measurement at the end of these experiments, we also decided to perform single-cell imaging experiments looking at the magnitude of the [Ca2+]c peak induced by caffeine after several K+ pulses in the presence of ryanodine. Fig 7 a shows that the [Ca2+]c increase induced by caffeine was little modified after five consecutive 30-s K+ pulses given in the presence of ryanodine (compare with the initial three caffeine additions). It is interesting to note that the second caffeine addition after the K+ + ryanodine pulses produced no [Ca2+]c increase, even though caffeine was always added in the absence of ryanodine. In fact, perfusion with ryanodine for a short period (30–60 s) before any caffeine addition was effective in promoting the typical use-dependent inhibition of ER refilling on application of caffeine (without ryanodine) 30–60 min later. This means that, even though ryanodine by itself does not produce any apparent effect on ER filling, it remains within the cells after washing and acts later, on stimulation of Ca2+ release (see below). Depolarization can also be produced using the nicotinic acetylcholine agonist DMPP instead of K+. Again here, consecutive pulses of 10 μM DMPP in the presence of ryanodine did not have any effect on a later [Ca2+]c peak induced by caffeine (data not shown). An alternative and perhaps more physiological depolarizing maneuver is field electric stimulation. Fig. 7 b shows the effect of several consecutive 10-s pulses at 10 Hz, before and after the addition of ryanodine. Again, we can observe that 5 mM caffeine produced the same [Ca2+]c peak after and before field electric stimulation. As in Fig. 7 a, consecutive additions of caffeine produced inhibition long after washing of ryanodine. The lack of effect of ryanodine in these experiments may be attributed to the much smaller activation of Ca2+ release by high K+-induced Ca2+ entry compared with that induced by caffeine.

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