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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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Effect of ER Ca2+ store depletion on FCCP-induced [Ca2+]i increase. (A) Fura-2–loaded acinar cells were stimulated with CCh in nominally Ca2+-free bath solution. After initiation of [Ca2+]i oscillations, treatment with cyclopiazonic acid resulted in a substantial increase in [Ca2+]i resulting from depletion of the ER Ca2+ store. After the return of [Ca2+]i to basal levels, treatment with FCCP resulted in no significant increase in [Ca2+]i. (B) Stimulation of fura-2–loaded acinar cells with high doses of CCh in nominally Ca2+-free bath solution resulted in a substantial increase in [Ca2+]i and depletion of intracellular Ca2+ pools. After the return of [Ca2+]i to basal levels, removal and subsequent reapplication of CCh resulted in no additional [Ca2+]i increase. Treatment with FCCP after stimulation with high-dose agonist resulted in no significant [Ca2+]i increase.
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Figure 2: Effect of ER Ca2+ store depletion on FCCP-induced [Ca2+]i increase. (A) Fura-2–loaded acinar cells were stimulated with CCh in nominally Ca2+-free bath solution. After initiation of [Ca2+]i oscillations, treatment with cyclopiazonic acid resulted in a substantial increase in [Ca2+]i resulting from depletion of the ER Ca2+ store. After the return of [Ca2+]i to basal levels, treatment with FCCP resulted in no significant increase in [Ca2+]i. (B) Stimulation of fura-2–loaded acinar cells with high doses of CCh in nominally Ca2+-free bath solution resulted in a substantial increase in [Ca2+]i and depletion of intracellular Ca2+ pools. After the return of [Ca2+]i to basal levels, removal and subsequent reapplication of CCh resulted in no additional [Ca2+]i increase. Treatment with FCCP after stimulation with high-dose agonist resulted in no significant [Ca2+]i increase.

Mentions: Because simple diffusion of Ca2+ in the absence of mitochondrial import is unlikely to account for the global Ca2+ rise evoked by low agonist concentrations, we investigated whether a regenerative Ca2+ release mechanism, dependent on internal stores, was involved. Treatment of fura-2–loaded acinar cells with the SERCA pump inhibitor cyclopiazonic acid after stimulation with an oscillatory dose of CCh resulted in a slow release of Ca2+ from endoplasmic reticulum (ER) stores, followed by inhibition of oscillations and a return of [Ca2+]i to basal levels (Fig. 2 A). Subsequent application of FCCP to depolarize mitochondria resulted in only a small increase in [Ca2+]i when compared with the increase obtained in the presence of an intact ER store (10 ± 6 vs. 207 ± 90 nM; n = 6). Similarly, stimulation with 0.1 mM CCh in nominally Ca2+-free bath solution resulted in a large increase in [Ca2+]i that returned to basal levels over the course of several minutes (Fig. 2 B). CCh was removed, and then reapplied to ensure that the ER store was depleted. Subsequent depolarization of mitochondria resulted in only a small [Ca2+]i increase (∼16 nM; n = 2). This data suggests that the enhanced Ca2+ release after mitochondrial depolarization was due predominantly to Ca2+ release from ER stores, and not from the mitochondria.


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)

Effect of ER Ca2+ store depletion on FCCP-induced [Ca2+]i increase. (A) Fura-2–loaded acinar cells were stimulated with CCh in nominally Ca2+-free bath solution. After initiation of [Ca2+]i oscillations, treatment with cyclopiazonic acid resulted in a substantial increase in [Ca2+]i resulting from depletion of the ER Ca2+ store. After the return of [Ca2+]i to basal levels, treatment with FCCP resulted in no significant increase in [Ca2+]i. (B) Stimulation of fura-2–loaded acinar cells with high doses of CCh in nominally Ca2+-free bath solution resulted in a substantial increase in [Ca2+]i and depletion of intracellular Ca2+ pools. After the return of [Ca2+]i to basal levels, removal and subsequent reapplication of CCh resulted in no additional [Ca2+]i increase. Treatment with FCCP after stimulation with high-dose agonist resulted in no significant [Ca2+]i increase.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2230627&req=5

Figure 2: Effect of ER Ca2+ store depletion on FCCP-induced [Ca2+]i increase. (A) Fura-2–loaded acinar cells were stimulated with CCh in nominally Ca2+-free bath solution. After initiation of [Ca2+]i oscillations, treatment with cyclopiazonic acid resulted in a substantial increase in [Ca2+]i resulting from depletion of the ER Ca2+ store. After the return of [Ca2+]i to basal levels, treatment with FCCP resulted in no significant increase in [Ca2+]i. (B) Stimulation of fura-2–loaded acinar cells with high doses of CCh in nominally Ca2+-free bath solution resulted in a substantial increase in [Ca2+]i and depletion of intracellular Ca2+ pools. After the return of [Ca2+]i to basal levels, removal and subsequent reapplication of CCh resulted in no additional [Ca2+]i increase. Treatment with FCCP after stimulation with high-dose agonist resulted in no significant [Ca2+]i increase.
Mentions: Because simple diffusion of Ca2+ in the absence of mitochondrial import is unlikely to account for the global Ca2+ rise evoked by low agonist concentrations, we investigated whether a regenerative Ca2+ release mechanism, dependent on internal stores, was involved. Treatment of fura-2–loaded acinar cells with the SERCA pump inhibitor cyclopiazonic acid after stimulation with an oscillatory dose of CCh resulted in a slow release of Ca2+ from endoplasmic reticulum (ER) stores, followed by inhibition of oscillations and a return of [Ca2+]i to basal levels (Fig. 2 A). Subsequent application of FCCP to depolarize mitochondria resulted in only a small increase in [Ca2+]i when compared with the increase obtained in the presence of an intact ER store (10 ± 6 vs. 207 ± 90 nM; n = 6). Similarly, stimulation with 0.1 mM CCh in nominally Ca2+-free bath solution resulted in a large increase in [Ca2+]i that returned to basal levels over the course of several minutes (Fig. 2 B). CCh was removed, and then reapplied to ensure that the ER store was depleted. Subsequent depolarization of mitochondria resulted in only a small [Ca2+]i increase (∼16 nM; n = 2). This data suggests that the enhanced Ca2+ release after mitochondrial depolarization was due predominantly to Ca2+ release from ER stores, and not from the mitochondria.

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