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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 mitochondrial depolarization on agonist-induced [Ca2+]i oscillations. (A) [Ca2+]i oscillations elicited by CCh were converted to an enhanced, sustained rise in [Ca2+]i after treatment with FCCP in fura-2–loaded acinar cells. As shown in the inset, treatment of TMRE-loaded acinar cells with FCCP caused a rapid decrease in TMRE fluorescence, indicative of mitochondrial depolarization. (B) Oscillations initiated by cholecystokinin were likewise converted to a sustained rise in [Ca2+]i after treatment with FCCP and the ATP synthase inhibitor oligomycin. (C) The respiratory chain inhibitor antimycin in combination with oligomycin similarly resulted in the conversion of oscillations to a sustained rise in [Ca2+]i. In all experiments, cells were maintained in nominally Ca2+-free bath solution throughout.
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Figure 1: Effect of mitochondrial depolarization on agonist-induced [Ca2+]i oscillations. (A) [Ca2+]i oscillations elicited by CCh were converted to an enhanced, sustained rise in [Ca2+]i after treatment with FCCP in fura-2–loaded acinar cells. As shown in the inset, treatment of TMRE-loaded acinar cells with FCCP caused a rapid decrease in TMRE fluorescence, indicative of mitochondrial depolarization. (B) Oscillations initiated by cholecystokinin were likewise converted to a sustained rise in [Ca2+]i after treatment with FCCP and the ATP synthase inhibitor oligomycin. (C) The respiratory chain inhibitor antimycin in combination with oligomycin similarly resulted in the conversion of oscillations to a sustained rise in [Ca2+]i. In all experiments, cells were maintained in nominally Ca2+-free bath solution throughout.

Mentions: Stimulation of acinar cells with low doses of the muscarinic agonist carbachol (CCh) (50–250 nM) produced oscillations in [Ca2+]i with a regular frequency (three to six per minute) and amplitude (100–300 nM) in nominally Ca2+-free external solution. The oscillations were generally maintained for up to 6 min (Yule and Gallacher 1988). Treatment with 0.5 μM FCCP, to reduce the driving force for mitochondrial import, resulted in an enhanced and sustained rise in [Ca2+]i lasting several hundred seconds that averaged 207 ± 90 nM above levels reached with agonist (Fig. 1 A, n = 10, and see Tinel et al. 1999). After washout of FCCP, some cells resumed oscillations in the continued presence of agonist (data not shown). Similar results were obtained using a range of FCCP concentrations (0.15–0.5 μM). Since inhibition of mitochondria could potentially result in local ATP depletion, additional experiments were performed combining mitochondrial inhibitors with the ATP synthase inhibitor oligomycin. This maneuver was designed to prevent the consumption of ATP by the synthase acting in reverse after dissipation of the mitochondrial membrane potential. As shown in Fig. 1 B, treatment with oligomycin (0.5 μM) did not alter the increase in [Ca2+]i evoked by FCCP (n = 3). Moreover, the ability of FCCP to convert an oscillatory into sustained [Ca2+]i increase was not agonist-specific, as the rise was observed in cells stimulated with either CCh (Fig. 1 A) or cholecystokinin (B). Additionally, treatment with the respiratory chain inhibitor antimycin (0.5 μM) in combination with 0.5 μM oligomycin resulted in a sustained [Ca2+]i rise to 273 ± 33 nM after CCh stimulation (Fig. 1 C, n = 8). Treatment with FCCP resulted in mitochondrial depolarization, as indicated by the mitochondrial membrane potential–sensitive dye, TMRE. TMRE fluorescence was concentrated in a well-defined region surrounding the apical pole. In cells loaded with 100 nM TMRE, a rapid decrease in fluorescence was observed after treatment with 0.5 μM FCCP (Fig. 1, inset, n = 3).


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 mitochondrial depolarization on agonist-induced [Ca2+]i oscillations. (A) [Ca2+]i oscillations elicited by CCh were converted to an enhanced, sustained rise in [Ca2+]i after treatment with FCCP in fura-2–loaded acinar cells. As shown in the inset, treatment of TMRE-loaded acinar cells with FCCP caused a rapid decrease in TMRE fluorescence, indicative of mitochondrial depolarization. (B) Oscillations initiated by cholecystokinin were likewise converted to a sustained rise in [Ca2+]i after treatment with FCCP and the ATP synthase inhibitor oligomycin. (C) The respiratory chain inhibitor antimycin in combination with oligomycin similarly resulted in the conversion of oscillations to a sustained rise in [Ca2+]i. In all experiments, cells were maintained in nominally Ca2+-free bath solution throughout.
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Related In: Results  -  Collection

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Figure 1: Effect of mitochondrial depolarization on agonist-induced [Ca2+]i oscillations. (A) [Ca2+]i oscillations elicited by CCh were converted to an enhanced, sustained rise in [Ca2+]i after treatment with FCCP in fura-2–loaded acinar cells. As shown in the inset, treatment of TMRE-loaded acinar cells with FCCP caused a rapid decrease in TMRE fluorescence, indicative of mitochondrial depolarization. (B) Oscillations initiated by cholecystokinin were likewise converted to a sustained rise in [Ca2+]i after treatment with FCCP and the ATP synthase inhibitor oligomycin. (C) The respiratory chain inhibitor antimycin in combination with oligomycin similarly resulted in the conversion of oscillations to a sustained rise in [Ca2+]i. In all experiments, cells were maintained in nominally Ca2+-free bath solution throughout.
Mentions: Stimulation of acinar cells with low doses of the muscarinic agonist carbachol (CCh) (50–250 nM) produced oscillations in [Ca2+]i with a regular frequency (three to six per minute) and amplitude (100–300 nM) in nominally Ca2+-free external solution. The oscillations were generally maintained for up to 6 min (Yule and Gallacher 1988). Treatment with 0.5 μM FCCP, to reduce the driving force for mitochondrial import, resulted in an enhanced and sustained rise in [Ca2+]i lasting several hundred seconds that averaged 207 ± 90 nM above levels reached with agonist (Fig. 1 A, n = 10, and see Tinel et al. 1999). After washout of FCCP, some cells resumed oscillations in the continued presence of agonist (data not shown). Similar results were obtained using a range of FCCP concentrations (0.15–0.5 μM). Since inhibition of mitochondria could potentially result in local ATP depletion, additional experiments were performed combining mitochondrial inhibitors with the ATP synthase inhibitor oligomycin. This maneuver was designed to prevent the consumption of ATP by the synthase acting in reverse after dissipation of the mitochondrial membrane potential. As shown in Fig. 1 B, treatment with oligomycin (0.5 μM) did not alter the increase in [Ca2+]i evoked by FCCP (n = 3). Moreover, the ability of FCCP to convert an oscillatory into sustained [Ca2+]i increase was not agonist-specific, as the rise was observed in cells stimulated with either CCh (Fig. 1 A) or cholecystokinin (B). Additionally, treatment with the respiratory chain inhibitor antimycin (0.5 μM) in combination with 0.5 μM oligomycin resulted in a sustained [Ca2+]i rise to 273 ± 33 nM after CCh stimulation (Fig. 1 C, n = 8). Treatment with FCCP resulted in mitochondrial depolarization, as indicated by the mitochondrial membrane potential–sensitive dye, TMRE. TMRE fluorescence was concentrated in a well-defined region surrounding the apical pole. In cells loaded with 100 nM TMRE, a rapid decrease in fluorescence was observed after treatment with 0.5 μM FCCP (Fig. 1, inset, n = 3).

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