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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 RyR inhibition on FCCP induced [Ca2+]i increase. Fura-2–loaded acinar cells were stimulated with an oscillatory dose of CCh in nominally Ca2+-free bath solution. After application of 100 μM ryanodine, cells were treated with FCCP to depolarize mitochondria. With RyR blocked, mitochondrial depolarization resulted in no significant enhancement in [Ca2+]i. As shown in the inset, treatment with ryanodine did not prevent mitochondrial depolarization as seen by the decrease in TMRE fluorescence after treatment with FCCP.
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Figure 4: Effect of RyR inhibition on FCCP induced [Ca2+]i increase. Fura-2–loaded acinar cells were stimulated with an oscillatory dose of CCh in nominally Ca2+-free bath solution. After application of 100 μM ryanodine, cells were treated with FCCP to depolarize mitochondria. With RyR blocked, mitochondrial depolarization resulted in no significant enhancement in [Ca2+]i. As shown in the inset, treatment with ryanodine did not prevent mitochondrial depolarization as seen by the decrease in TMRE fluorescence after treatment with FCCP.

Mentions: To determine the functional significance of this colocalization, the contribution of RyR to the FCCP-induced enhancement of [Ca2+]i was investigated. After stimulation with CCh, cells were superfused with 100 μM ryanodine, a concentration that has been reported to block RyR in a nonconducting state (Lai et al. 1989; McGrew et al. 1989; for review see Shoshan-Barmatz and Ashley 1998). Ryanodine treatment significantly decreased the amplitude of CCh-induced oscillations from 157 ± 25 to 96 ± 21 nM above basal (Fig. 4, n = 5, P = 0.003), but did not significantly alter the frequency of oscillations. Moreover, in the presence of ryanodine, FCCP treatment failed to evoke an additional [Ca2+]i rise. Measurement of TMRE fluorescence showed that treatment with ryanodine did not affect the ability of FCCP to depolarize mitochondria (Fig. 4, inset, n = 5). These data indicate that ryanodine treatment disrupts a functional interaction between mitochondria and ryanodine-sensitive Ca2+ stores.


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 RyR inhibition on FCCP induced [Ca2+]i increase. Fura-2–loaded acinar cells were stimulated with an oscillatory dose of CCh in nominally Ca2+-free bath solution. After application of 100 μM ryanodine, cells were treated with FCCP to depolarize mitochondria. With RyR blocked, mitochondrial depolarization resulted in no significant enhancement in [Ca2+]i. As shown in the inset, treatment with ryanodine did not prevent mitochondrial depolarization as seen by the decrease in TMRE fluorescence after treatment with FCCP.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Effect of RyR inhibition on FCCP induced [Ca2+]i increase. Fura-2–loaded acinar cells were stimulated with an oscillatory dose of CCh in nominally Ca2+-free bath solution. After application of 100 μM ryanodine, cells were treated with FCCP to depolarize mitochondria. With RyR blocked, mitochondrial depolarization resulted in no significant enhancement in [Ca2+]i. As shown in the inset, treatment with ryanodine did not prevent mitochondrial depolarization as seen by the decrease in TMRE fluorescence after treatment with FCCP.
Mentions: To determine the functional significance of this colocalization, the contribution of RyR to the FCCP-induced enhancement of [Ca2+]i was investigated. After stimulation with CCh, cells were superfused with 100 μM ryanodine, a concentration that has been reported to block RyR in a nonconducting state (Lai et al. 1989; McGrew et al. 1989; for review see Shoshan-Barmatz and Ashley 1998). Ryanodine treatment significantly decreased the amplitude of CCh-induced oscillations from 157 ± 25 to 96 ± 21 nM above basal (Fig. 4, n = 5, P = 0.003), but did not significantly alter the frequency of oscillations. Moreover, in the presence of ryanodine, FCCP treatment failed to evoke an additional [Ca2+]i rise. Measurement of TMRE fluorescence showed that treatment with ryanodine did not affect the ability of FCCP to depolarize mitochondria (Fig. 4, inset, n = 5). These data indicate that ryanodine treatment disrupts a functional interaction between mitochondria and ryanodine-sensitive Ca2+ stores.

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