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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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Subcellular localization of RyRs and mitochondria in acinar cells. A and C show bright-field images of clusters of acinar cells that correspond to the confocal fluorescence images in B and D, respectively. (B) An acinus stained with BODIPY-ryanodine to visualize RyR. Diffuse labeling is seen throughout the cytoplasm; however, the boundary between the zymogen granule containing region and the basal portion of the cell exhibits the greatest intensity of labeling. (D) An acinus stained with Mitotracker red to visualize mitochondria. The majority of mitochondria are present in a similar location to RyR, surrounding the apical, granule-containing region of the cell.
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Figure 3: Subcellular localization of RyRs and mitochondria in acinar cells. A and C show bright-field images of clusters of acinar cells that correspond to the confocal fluorescence images in B and D, respectively. (B) An acinus stained with BODIPY-ryanodine to visualize RyR. Diffuse labeling is seen throughout the cytoplasm; however, the boundary between the zymogen granule containing region and the basal portion of the cell exhibits the greatest intensity of labeling. (D) An acinus stained with Mitotracker red to visualize mitochondria. The majority of mitochondria are present in a similar location to RyR, surrounding the apical, granule-containing region of the cell.

Mentions: Work by Leite et al. 1999 demonstrated the presence of type 2 RyR in acinar cells, consistent with the possibility that the global [Ca2+]i increase observed after mitochondrial depolarization was dependent on RyR. We therefore investigated whether RyR and mitochondria might be morphologically and/or functionally linked. Confocal microscopy revealed that the BODIPY-ryanodine fluorescence was concentrated in a region surrounding the apical pole of the cell (Fig. 3A and Fig. B; n = 5 preparations). More diffuse fluorescence was seen throughout the apical and basal regions, while no fluorescence was seen when cells were incubated with a 50-fold excess of nonfluorescent ryanodine before incubation with BODIPY-ryanodine (n = 2 preparations). Mitochondrial distribution was investigated using the mitochondrial-specific dye Mitotracker red. Mitochondria were found to be concentrated in a similar region, surrounding the apical zymogen granule-containing region (Fig. 3C and Fig. D, and see Tinel et al. 1999). This distribution was similar to that observed with TMRE. The distribution of RyR and mitochondria thus overlaps in acinar cells.


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)

Subcellular localization of RyRs and mitochondria in acinar cells. A and C show bright-field images of clusters of acinar cells that correspond to the confocal fluorescence images in B and D, respectively. (B) An acinus stained with BODIPY-ryanodine to visualize RyR. Diffuse labeling is seen throughout the cytoplasm; however, the boundary between the zymogen granule containing region and the basal portion of the cell exhibits the greatest intensity of labeling. (D) An acinus stained with Mitotracker red to visualize mitochondria. The majority of mitochondria are present in a similar location to RyR, surrounding the apical, granule-containing region of the cell.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Subcellular localization of RyRs and mitochondria in acinar cells. A and C show bright-field images of clusters of acinar cells that correspond to the confocal fluorescence images in B and D, respectively. (B) An acinus stained with BODIPY-ryanodine to visualize RyR. Diffuse labeling is seen throughout the cytoplasm; however, the boundary between the zymogen granule containing region and the basal portion of the cell exhibits the greatest intensity of labeling. (D) An acinus stained with Mitotracker red to visualize mitochondria. The majority of mitochondria are present in a similar location to RyR, surrounding the apical, granule-containing region of the cell.
Mentions: Work by Leite et al. 1999 demonstrated the presence of type 2 RyR in acinar cells, consistent with the possibility that the global [Ca2+]i increase observed after mitochondrial depolarization was dependent on RyR. We therefore investigated whether RyR and mitochondria might be morphologically and/or functionally linked. Confocal microscopy revealed that the BODIPY-ryanodine fluorescence was concentrated in a region surrounding the apical pole of the cell (Fig. 3A and Fig. B; n = 5 preparations). More diffuse fluorescence was seen throughout the apical and basal regions, while no fluorescence was seen when cells were incubated with a 50-fold excess of nonfluorescent ryanodine before incubation with BODIPY-ryanodine (n = 2 preparations). Mitochondrial distribution was investigated using the mitochondrial-specific dye Mitotracker red. Mitochondria were found to be concentrated in a similar region, surrounding the apical zymogen granule-containing region (Fig. 3C and Fig. D, and see Tinel et al. 1999). This distribution was similar to that observed with TMRE. The distribution of RyR and mitochondria thus overlaps in acinar cells.

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