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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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Photolytic release of InsP3 causes propagation of a global [Ca2+]i wave after mitochondrial depolarization and RyR activation. (A, I and III) [Ca2+]i traces and OGB-2 fluorescence images showing the [Ca2+]i increase after photolysis of 1 μM caged InsP3 at the time indicated by the arrow under control conditions. The [Ca2+]i increase initiated at the apical trigger zone and remained apically localized. [Ca2+]i traces were generated from the apical and basal regions depicted in the brightfield image (red, apical; black, basal). (II and IV) Second uncaging under control conditions illustrates that the initiation and localization of the [Ca2+]i change are reproducible after repeated photolytic events. (B, I and III) Photolytic release of 1 μM caged InsP3 under control conditions evokes an apically localized [Ca2+]i increase, as shown in the [Ca2+]i traces and OGB-2 fluorescence images. The [Ca2+]i traces were generated from the apical and basal regions depicted in the brightfield image. (II and IV) After treatment with FCCP for 3 min, uncaging evokes a global rise in [Ca2+]i that initiates at the apical trigger zone and propagates through the cell as a [Ca2+]i wave. (C, I and III) Photolysis of 1 μM caged InsP3 under control conditions with RyR inhibited evokes an apically localized [Ca2+]i increase, as shown in the [Ca2+]i traces and OGB2 fluorescence images. The [Ca2+]i traces were generated from the apical and basal regions depicted in the brightfield image. (II and IV) After treatment with FCCP for 3 min, uncaging evokes an apically localized [Ca2+]i increase that does not spread significantly into the basal region of the cell.
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Figure 6: Photolytic release of InsP3 causes propagation of a global [Ca2+]i wave after mitochondrial depolarization and RyR activation. (A, I and III) [Ca2+]i traces and OGB-2 fluorescence images showing the [Ca2+]i increase after photolysis of 1 μM caged InsP3 at the time indicated by the arrow under control conditions. The [Ca2+]i increase initiated at the apical trigger zone and remained apically localized. [Ca2+]i traces were generated from the apical and basal regions depicted in the brightfield image (red, apical; black, basal). (II and IV) Second uncaging under control conditions illustrates that the initiation and localization of the [Ca2+]i change are reproducible after repeated photolytic events. (B, I and III) Photolytic release of 1 μM caged InsP3 under control conditions evokes an apically localized [Ca2+]i increase, as shown in the [Ca2+]i traces and OGB-2 fluorescence images. The [Ca2+]i traces were generated from the apical and basal regions depicted in the brightfield image. (II and IV) After treatment with FCCP for 3 min, uncaging evokes a global rise in [Ca2+]i that initiates at the apical trigger zone and propagates through the cell as a [Ca2+]i wave. (C, I and III) Photolysis of 1 μM caged InsP3 under control conditions with RyR inhibited evokes an apically localized [Ca2+]i increase, as shown in the [Ca2+]i traces and OGB2 fluorescence images. The [Ca2+]i traces were generated from the apical and basal regions depicted in the brightfield image. (II and IV) After treatment with FCCP for 3 min, uncaging evokes an apically localized [Ca2+]i increase that does not spread significantly into the basal region of the cell.

Mentions: Since pharmacological inhibition of mitochondria revealed what appeared to be CICR from a ryanodine-sensitive store, we investigated whether this Ca2+ release was physiologically relevant to [Ca2+]i wave propagation in acinar cells. Photolytic release of low levels of InsP3 was used to evoke [Ca2+]i increases that remained largely confined to the apical region of whole cell patch clamped cells. The spatial characteristics of the [Ca2+]i signal were monitored by digital imaging of OGB-2 fluorescence. This approach allowed the effects of mitochondrial depolarization and RyR blockade on the properties of a propagating [Ca2+]i wave to be directly investigated. On average, a single UV flash generated a reproducible, local [Ca2+]i signal that traveled 7.7 ± 0.8 μm from the site of initiation at a speed of 16.0 ± 0.1 μm/s (Fig. 6 A, n = 8). This type of “contained” signal transiently raised [Ca2+]i within the apical half of the cell (cell diameter = 16.5 ± 0.4 μm; n = 36). As shown in Fig. 6 B, I and III, photolysis of 1 μM caged InsP3 evoked a localized [Ca2+]i increase that, after treatment with FCCP, still initiated at the trigger zone, but subsequently spread throughout the cell. On average, the [Ca2+]i signal now traveled 15.6 ± 1.3 μm (Fig. 6 B, II and IV, n = 4), consistent with the hypothesis that mitochondrial buffering is important for restricting this type of [Ca2+]i signal. Next, we repeated these experiments after treatment with ryanodine to test the hypothesis that Ca2+ release from RyR contributed to the propagation of a [Ca2+]i wave throughout the cell. Photolysis of 1 μM caged InsP3 in the presence of 100 μM ryanodine evoked a localized [Ca2+]i increase, similar to that produced by the control uncaging in the absence of ryanodine (Fig. 6 C, I and III). However, a subsequent flash after treatment with FCCP now failed to evoke a global elevation in [Ca2+]i, the signal propagating only 6.3 ± 2.1 μm, significantly different to the distance observed in FCCP alone (Fig. 6 C, II and IV, n = 3, P = 0.008). The observation that Ca2+ did not spread throughout the cell after mitochondrial depolarization when RyRs were inhibited suggests that RyRs play a central role in propagating [Ca2+]i increases throughout the cell and points to a potential role for mitochondria in modulating RyR activation.


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)

Photolytic release of InsP3 causes propagation of a global [Ca2+]i wave after mitochondrial depolarization and RyR activation. (A, I and III) [Ca2+]i traces and OGB-2 fluorescence images showing the [Ca2+]i increase after photolysis of 1 μM caged InsP3 at the time indicated by the arrow under control conditions. The [Ca2+]i increase initiated at the apical trigger zone and remained apically localized. [Ca2+]i traces were generated from the apical and basal regions depicted in the brightfield image (red, apical; black, basal). (II and IV) Second uncaging under control conditions illustrates that the initiation and localization of the [Ca2+]i change are reproducible after repeated photolytic events. (B, I and III) Photolytic release of 1 μM caged InsP3 under control conditions evokes an apically localized [Ca2+]i increase, as shown in the [Ca2+]i traces and OGB-2 fluorescence images. The [Ca2+]i traces were generated from the apical and basal regions depicted in the brightfield image. (II and IV) After treatment with FCCP for 3 min, uncaging evokes a global rise in [Ca2+]i that initiates at the apical trigger zone and propagates through the cell as a [Ca2+]i wave. (C, I and III) Photolysis of 1 μM caged InsP3 under control conditions with RyR inhibited evokes an apically localized [Ca2+]i increase, as shown in the [Ca2+]i traces and OGB2 fluorescence images. The [Ca2+]i traces were generated from the apical and basal regions depicted in the brightfield image. (II and IV) After treatment with FCCP for 3 min, uncaging evokes an apically localized [Ca2+]i increase that does not spread significantly into the basal region of the cell.
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Figure 6: Photolytic release of InsP3 causes propagation of a global [Ca2+]i wave after mitochondrial depolarization and RyR activation. (A, I and III) [Ca2+]i traces and OGB-2 fluorescence images showing the [Ca2+]i increase after photolysis of 1 μM caged InsP3 at the time indicated by the arrow under control conditions. The [Ca2+]i increase initiated at the apical trigger zone and remained apically localized. [Ca2+]i traces were generated from the apical and basal regions depicted in the brightfield image (red, apical; black, basal). (II and IV) Second uncaging under control conditions illustrates that the initiation and localization of the [Ca2+]i change are reproducible after repeated photolytic events. (B, I and III) Photolytic release of 1 μM caged InsP3 under control conditions evokes an apically localized [Ca2+]i increase, as shown in the [Ca2+]i traces and OGB-2 fluorescence images. The [Ca2+]i traces were generated from the apical and basal regions depicted in the brightfield image. (II and IV) After treatment with FCCP for 3 min, uncaging evokes a global rise in [Ca2+]i that initiates at the apical trigger zone and propagates through the cell as a [Ca2+]i wave. (C, I and III) Photolysis of 1 μM caged InsP3 under control conditions with RyR inhibited evokes an apically localized [Ca2+]i increase, as shown in the [Ca2+]i traces and OGB2 fluorescence images. The [Ca2+]i traces were generated from the apical and basal regions depicted in the brightfield image. (II and IV) After treatment with FCCP for 3 min, uncaging evokes an apically localized [Ca2+]i increase that does not spread significantly into the basal region of the cell.
Mentions: Since pharmacological inhibition of mitochondria revealed what appeared to be CICR from a ryanodine-sensitive store, we investigated whether this Ca2+ release was physiologically relevant to [Ca2+]i wave propagation in acinar cells. Photolytic release of low levels of InsP3 was used to evoke [Ca2+]i increases that remained largely confined to the apical region of whole cell patch clamped cells. The spatial characteristics of the [Ca2+]i signal were monitored by digital imaging of OGB-2 fluorescence. This approach allowed the effects of mitochondrial depolarization and RyR blockade on the properties of a propagating [Ca2+]i wave to be directly investigated. On average, a single UV flash generated a reproducible, local [Ca2+]i signal that traveled 7.7 ± 0.8 μm from the site of initiation at a speed of 16.0 ± 0.1 μm/s (Fig. 6 A, n = 8). This type of “contained” signal transiently raised [Ca2+]i within the apical half of the cell (cell diameter = 16.5 ± 0.4 μm; n = 36). As shown in Fig. 6 B, I and III, photolysis of 1 μM caged InsP3 evoked a localized [Ca2+]i increase that, after treatment with FCCP, still initiated at the trigger zone, but subsequently spread throughout the cell. On average, the [Ca2+]i signal now traveled 15.6 ± 1.3 μm (Fig. 6 B, II and IV, n = 4), consistent with the hypothesis that mitochondrial buffering is important for restricting this type of [Ca2+]i signal. Next, we repeated these experiments after treatment with ryanodine to test the hypothesis that Ca2+ release from RyR contributed to the propagation of a [Ca2+]i wave throughout the cell. Photolysis of 1 μM caged InsP3 in the presence of 100 μM ryanodine evoked a localized [Ca2+]i increase, similar to that produced by the control uncaging in the absence of ryanodine (Fig. 6 C, I and III). However, a subsequent flash after treatment with FCCP now failed to evoke a global elevation in [Ca2+]i, the signal propagating only 6.3 ± 2.1 μm, significantly different to the distance observed in FCCP alone (Fig. 6 C, II and IV, n = 3, P = 0.008). The observation that Ca2+ did not spread throughout the cell after mitochondrial depolarization when RyRs were inhibited suggests that RyRs play a central role in propagating [Ca2+]i increases throughout the cell and points to a potential role for mitochondria in modulating RyR activation.

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