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Calcium wave propagation in pancreatic acinar cells: functional interaction of inositol 1,4,5-trisphosphate receptors, ryanodine receptors, and mitochondria.

Straub SV, Giovannucci DR, Yule DI - J. Gen. Physiol. (2000)

Bottom Line: Similarly, "uncaging" of physiological [Ca(2+)](i) levels in whole-cell patch-clamped cells resulted in rapid activation of a Ca(2+)-activated current, the recovery of which was prolonged by inhibition of mitochondrial import.This effect was also abolished by ryanodine receptor (RyR) blockade.Global [Ca(2+)](i) rises initiated by InsP(3) were also reduced by ryanodine, limiting the increase to a region slightly larger than the trigger zone.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Physiology, University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642, USA.

ABSTRACT
In pancreatic acinar cells, inositol 1,4,5-trisphosphate (InsP(3))-dependent cytosolic calcium ([Ca(2+)](i)) increases resulting from agonist stimulation are initiated in an apical "trigger zone," where the vast majority of InsP(3) receptors (InsP(3)R) are localized. At threshold stimulation, [Ca(2+)](i) signals are confined to this region, whereas at concentrations of agonists that optimally evoke secretion, a global Ca(2+) wave results. Simple diffusion of Ca(2+) from the trigger zone is unlikely to account for a global [Ca(2+)](i) elevation. Furthermore, mitochondrial import has been reported to limit Ca(2+) diffusion from the trigger zone. As such, there is no consensus as to how local [Ca(2+)](i) signals become global responses. This study therefore investigated the mechanism responsible for these events. Agonist-evoked [Ca(2+)](i) oscillations were converted to sustained [Ca(2+)](i) increases after inhibition of mitochondrial Ca(2+) import. These [Ca(2+)](i) increases were dependent on Ca(2+) release from the endoplasmic reticulum and were blocked by 100 microM ryanodine. Similarly, "uncaging" of physiological [Ca(2+)](i) levels in whole-cell patch-clamped cells resulted in rapid activation of a Ca(2+)-activated current, the recovery of which was prolonged by inhibition of mitochondrial import. This effect was also abolished by ryanodine receptor (RyR) blockade. Photolysis of d-myo InsP(3) P(4(5))-1-(2-nitrophenyl)-ethyl ester (caged InsP(3)) produced either apically localized or global [Ca(2+)](i) increases in a dose-dependent manner, as visualized by digital imaging. Mitochondrial inhibition permitted apically localized increases to propagate throughout the cell as a wave, but this propagation was inhibited by ryanodine and was not seen for minimal control responses resembling [Ca(2+)](i) puffs. Global [Ca(2+)](i) rises initiated by InsP(3) were also reduced by ryanodine, limiting the increase to a region slightly larger than the trigger zone. These data suggest that, while Ca(2+) release is initially triggered through InsP(3)R, release by RyRs is the dominant mechanism for propagating global waves. In addition, mitochondrial Ca(2+) import controls the spread of Ca(2+) throughout acinar cells by modulating RyR activation.

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Photolysis of caged Ca2+ evokes CICR after mitochondrial depolarization that is dependent upon RyR activation. (A) Rapid, global [Ca2+]i increases were evoked by flash photolysis of NP-EGTA. (B) After photolysis to evoke a control current, treatment with FCCP for 3 min followed by photolysis produced a current that was significantly delayed in the time to recovery (Trec). (C) After RyR inhibition, the current produced by photolysis after a 3-min treatment with FCCP did not differ significantly compared with control. (D) Pooled data from experiments: to normalize values, pooled data represents the current evoked after FCCP treatment minus the current evoked under control conditions for each experimental condition. Data represented as mean ± SEM. *Significant difference from control, P < 0.022; n = 7 for control and control + FCCP; n = 6 for ryanodine and ryanodine + FCCP.
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Figure 5: Photolysis of caged Ca2+ evokes CICR after mitochondrial depolarization that is dependent upon RyR activation. (A) Rapid, global [Ca2+]i increases were evoked by flash photolysis of NP-EGTA. (B) After photolysis to evoke a control current, treatment with FCCP for 3 min followed by photolysis produced a current that was significantly delayed in the time to recovery (Trec). (C) After RyR inhibition, the current produced by photolysis after a 3-min treatment with FCCP did not differ significantly compared with control. (D) Pooled data from experiments: to normalize values, pooled data represents the current evoked after FCCP treatment minus the current evoked under control conditions for each experimental condition. Data represented as mean ± SEM. *Significant difference from control, P < 0.022; n = 7 for control and control + FCCP; n = 6 for ryanodine and ryanodine + FCCP.

Mentions: The aforementioned experiments reveal that Ca2+ release through ryanodine-sensitive stores can be evoked after mitochondrial depolarization, and further suggest that this occurs as a result of colocalization. To determine whether Ca2+ in the vicinity of mitochondria could influence RyR activity, we evoked transient global elevations in [Ca2+]i through photolytic release of caged Ca2+ (Ellis-Davies and Kaplan 1994; Takahashi et al. 1999; Zahradnikova et al. 1999). Changes in [Ca2+]i were monitored in whole-cell patch-clamped acinar cells using a Ca2+-activated Cl− current. This current has been extensively used to report changes in [Ca2+]i in this cell type (Thorn et al. 1993; Cancela et al. 1999; Kidd et al. 1999; Park et al. 1999; Xu et al. 1999). Photolysis was achieved by a 0.5-ms UV flash 3 min after obtaining a stable whole-cell configuration, a time sufficient to allow equilibration between the pipette solution and cell. Repetitive uncagings at regular intervals evoked reproducible increases in the Ca2+-activated Cl− current (Fig. 5 A). Using the low-affinity Ca2+-sensitive dye benzothiazole coumarin, an in situ calibration (Ito et al. 1997) estimated that, on average, photolysis of NP-EGTA produced an increase in [Ca2+]i of 7 μM, a level within the range of [Ca2+]i increases in the apical region typically associated with high doses of agonist (Ito et al. 1997). Currents were evoked before and after FCCP treatment and the total charge, recovery time, and peak current values were respectively compared. After treatment with FCCP for 3 min, photolysis resulted in a current significantly enhanced with respect to the total charge passed (Qtot) (34,685 ± 12,070 pC vs. 8,735 ± 3,554 pC, Fig. 5 B; n = 7, P = 0.016). This increase in Qtot was the result of a significant, nearly fourfold increase in the time to steady state recovery (Trec) (214 ± 61 vs. 54 ± 12 s, n = 7, P = 0.022), since no significant increase in the peak current (Ipeak) was observed (612 ± 104 vs. 455 ± 59 pA, n = 7, P = 0.156). No measurable current change was produced by FCCP treatment in the absence of photolysis. Control currents evoked in the presence or absence of ryanodine in the pipette solution were not significantly different (Qtot = 8,814 ± 2,346 vs. 8,735 ± 3,554 pC, P = 0.628; Trec = 39 ± 6 vs. 53 ± 12 s, P = 0.234; n = 7 control, n = 6 ryanodine). In contrast, in the presence of ryanodine, the evoked currents in FCCP were significantly altered compared with FCCP treatment alone following subtraction of the control currents (5,353 ± 2,751 vs. 25,950 ± 8,987 pC, P = 0.008; 17 ± 6 vs. 160 ± 52 s, P = 0.022). The ability of ryanodine to abolish the prolonged Trec induced by FCCP suggests that the slow recovery time was due, at least in part, to persistent Ca2+ release from a Ca2+- and ryanodine-sensitive store.


Calcium wave propagation in pancreatic acinar cells: functional interaction of inositol 1,4,5-trisphosphate receptors, ryanodine receptors, and mitochondria.

Straub SV, Giovannucci DR, Yule DI - J. Gen. Physiol. (2000)

Photolysis of caged Ca2+ evokes CICR after mitochondrial depolarization that is dependent upon RyR activation. (A) Rapid, global [Ca2+]i increases were evoked by flash photolysis of NP-EGTA. (B) After photolysis to evoke a control current, treatment with FCCP for 3 min followed by photolysis produced a current that was significantly delayed in the time to recovery (Trec). (C) After RyR inhibition, the current produced by photolysis after a 3-min treatment with FCCP did not differ significantly compared with control. (D) Pooled data from experiments: to normalize values, pooled data represents the current evoked after FCCP treatment minus the current evoked under control conditions for each experimental condition. Data represented as mean ± SEM. *Significant difference from control, P < 0.022; n = 7 for control and control + FCCP; n = 6 for ryanodine and ryanodine + FCCP.
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Related In: Results  -  Collection

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Figure 5: Photolysis of caged Ca2+ evokes CICR after mitochondrial depolarization that is dependent upon RyR activation. (A) Rapid, global [Ca2+]i increases were evoked by flash photolysis of NP-EGTA. (B) After photolysis to evoke a control current, treatment with FCCP for 3 min followed by photolysis produced a current that was significantly delayed in the time to recovery (Trec). (C) After RyR inhibition, the current produced by photolysis after a 3-min treatment with FCCP did not differ significantly compared with control. (D) Pooled data from experiments: to normalize values, pooled data represents the current evoked after FCCP treatment minus the current evoked under control conditions for each experimental condition. Data represented as mean ± SEM. *Significant difference from control, P < 0.022; n = 7 for control and control + FCCP; n = 6 for ryanodine and ryanodine + FCCP.
Mentions: The aforementioned experiments reveal that Ca2+ release through ryanodine-sensitive stores can be evoked after mitochondrial depolarization, and further suggest that this occurs as a result of colocalization. To determine whether Ca2+ in the vicinity of mitochondria could influence RyR activity, we evoked transient global elevations in [Ca2+]i through photolytic release of caged Ca2+ (Ellis-Davies and Kaplan 1994; Takahashi et al. 1999; Zahradnikova et al. 1999). Changes in [Ca2+]i were monitored in whole-cell patch-clamped acinar cells using a Ca2+-activated Cl− current. This current has been extensively used to report changes in [Ca2+]i in this cell type (Thorn et al. 1993; Cancela et al. 1999; Kidd et al. 1999; Park et al. 1999; Xu et al. 1999). Photolysis was achieved by a 0.5-ms UV flash 3 min after obtaining a stable whole-cell configuration, a time sufficient to allow equilibration between the pipette solution and cell. Repetitive uncagings at regular intervals evoked reproducible increases in the Ca2+-activated Cl− current (Fig. 5 A). Using the low-affinity Ca2+-sensitive dye benzothiazole coumarin, an in situ calibration (Ito et al. 1997) estimated that, on average, photolysis of NP-EGTA produced an increase in [Ca2+]i of 7 μM, a level within the range of [Ca2+]i increases in the apical region typically associated with high doses of agonist (Ito et al. 1997). Currents were evoked before and after FCCP treatment and the total charge, recovery time, and peak current values were respectively compared. After treatment with FCCP for 3 min, photolysis resulted in a current significantly enhanced with respect to the total charge passed (Qtot) (34,685 ± 12,070 pC vs. 8,735 ± 3,554 pC, Fig. 5 B; n = 7, P = 0.016). This increase in Qtot was the result of a significant, nearly fourfold increase in the time to steady state recovery (Trec) (214 ± 61 vs. 54 ± 12 s, n = 7, P = 0.022), since no significant increase in the peak current (Ipeak) was observed (612 ± 104 vs. 455 ± 59 pA, n = 7, P = 0.156). No measurable current change was produced by FCCP treatment in the absence of photolysis. Control currents evoked in the presence or absence of ryanodine in the pipette solution were not significantly different (Qtot = 8,814 ± 2,346 vs. 8,735 ± 3,554 pC, P = 0.628; Trec = 39 ± 6 vs. 53 ± 12 s, P = 0.234; n = 7 control, n = 6 ryanodine). In contrast, in the presence of ryanodine, the evoked currents in FCCP were significantly altered compared with FCCP treatment alone following subtraction of the control currents (5,353 ± 2,751 vs. 25,950 ± 8,987 pC, P = 0.008; 17 ± 6 vs. 160 ± 52 s, P = 0.022). The ability of ryanodine to abolish the prolonged Trec induced by FCCP suggests that the slow recovery time was due, at least in part, to persistent Ca2+ release from a Ca2+- and ryanodine-sensitive store.

Bottom Line: Similarly, "uncaging" of physiological [Ca(2+)](i) levels in whole-cell patch-clamped cells resulted in rapid activation of a Ca(2+)-activated current, the recovery of which was prolonged by inhibition of mitochondrial import.This effect was also abolished by ryanodine receptor (RyR) blockade.Global [Ca(2+)](i) rises initiated by InsP(3) were also reduced by ryanodine, limiting the increase to a region slightly larger than the trigger zone.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Physiology, University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642, USA.

ABSTRACT
In pancreatic acinar cells, inositol 1,4,5-trisphosphate (InsP(3))-dependent cytosolic calcium ([Ca(2+)](i)) increases resulting from agonist stimulation are initiated in an apical "trigger zone," where the vast majority of InsP(3) receptors (InsP(3)R) are localized. At threshold stimulation, [Ca(2+)](i) signals are confined to this region, whereas at concentrations of agonists that optimally evoke secretion, a global Ca(2+) wave results. Simple diffusion of Ca(2+) from the trigger zone is unlikely to account for a global [Ca(2+)](i) elevation. Furthermore, mitochondrial import has been reported to limit Ca(2+) diffusion from the trigger zone. As such, there is no consensus as to how local [Ca(2+)](i) signals become global responses. This study therefore investigated the mechanism responsible for these events. Agonist-evoked [Ca(2+)](i) oscillations were converted to sustained [Ca(2+)](i) increases after inhibition of mitochondrial Ca(2+) import. These [Ca(2+)](i) increases were dependent on Ca(2+) release from the endoplasmic reticulum and were blocked by 100 microM ryanodine. Similarly, "uncaging" of physiological [Ca(2+)](i) levels in whole-cell patch-clamped cells resulted in rapid activation of a Ca(2+)-activated current, the recovery of which was prolonged by inhibition of mitochondrial import. This effect was also abolished by ryanodine receptor (RyR) blockade. Photolysis of d-myo InsP(3) P(4(5))-1-(2-nitrophenyl)-ethyl ester (caged InsP(3)) produced either apically localized or global [Ca(2+)](i) increases in a dose-dependent manner, as visualized by digital imaging. Mitochondrial inhibition permitted apically localized increases to propagate throughout the cell as a wave, but this propagation was inhibited by ryanodine and was not seen for minimal control responses resembling [Ca(2+)](i) puffs. Global [Ca(2+)](i) rises initiated by InsP(3) were also reduced by ryanodine, limiting the increase to a region slightly larger than the trigger zone. These data suggest that, while Ca(2+) release is initially triggered through InsP(3)R, release by RyRs is the dominant mechanism for propagating global waves. In addition, mitochondrial Ca(2+) import controls the spread of Ca(2+) throughout acinar cells by modulating RyR activation.

Show MeSH
Related in: MedlinePlus