Correspondence to: David R. Giovannucci, Department of Pharmacology and Physiology, University of Rochester, School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642. Fax:716-273-2652 E-mail:david_giovannucci{at}urmc.rochester.edu.
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Abstract |
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In pancreatic acinar cells, inositol 1,4,5-trisphosphate (InsP3)dependent cytosolic calcium ([Ca2+]i) increases resulting from agonist stimulation are initiated in an apical "trigger zone," where the vast majority of InsP3 receptors (InsP3R) are localized. At threshold stimulation, [Ca2+]i signals are confined to this region, whereas at concentrations of agonists that optimally evoke secretion, a global Ca2+ wave results. Simple diffusion of Ca2+ from the trigger zone is unlikely to account for a global [Ca2+]i elevation. Furthermore, mitochondrial import has been reported to limit Ca2+ diffusion from the trigger zone. As such, there is no consensus as to how local [Ca2+]i signals become global responses. This study therefore investigated the mechanism responsible for these events. Agonist-evoked [Ca2+]i oscillations were converted to sustained [Ca2+]i increases after inhibition of mitochondrial Ca2+ import. These [Ca2+]i increases were dependent on Ca2+ release from the endoplasmic reticulum and were blocked by 100 µM ryanodine. Similarly, "uncaging" of physiological [Ca2+]i levels in whole-cell patch-clamped cells resulted in rapid activation of a Ca2+-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 InsP3 P4(5)-1-(2-nitrophenyl)-ethyl ester (caged InsP3) produced either apically localized or global [Ca2+]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 [Ca2+]i puffs. Global [Ca2+]i rises initiated by InsP3 were also reduced by ryanodine, limiting the increase to a region slightly larger than the trigger zone. These data suggest that, while Ca2+ release is initially triggered through InsP3R, release by RyRs is the dominant mechanism for propagating global waves. In addition, mitochondrial Ca2+ import controls the spread of Ca2+ throughout acinar cells by modulating RyR activation.
Key Words: calcium dynamics, intracellular signaling, exocrine cells, flash photolysis, digital imaging
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INTRODUCTION |
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Ca2+ release from inositol 1,4,5-trisphosphate (InsP3)1sensitive stores exerts control over a wide variety of physiological processes including secretion, gene transcription, and apoptosis (
Since it is known that the initial trigger for global [Ca2+]i elevations is Ca2+ release from InsP3R in the apical trigger zone (
Much recent work has suggested that mitochondrial Ca2+ import (for review, see
The focus of this study was to investigate the mechanisms by which an initial, apically localized Ca2+ release event is subsequently propagated throughout the cell and the possible modulatory role of mitochondria in this process. We show that, after Ca2+ release from InsP3R, the propagation of Ca2+ waves is modulated by a functional interaction between RyR and mitochondria. This indicates that CICR from RyR is likely the mechanism by which an apical [Ca2+]i signal triggers a global [Ca2+]i wave.
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METHODS |
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Materials
Purified collagenase (CLSPA grade) was purchased from Worthington Biochemicals. Fura-2 AM was purchased from Teflabs. Oregon green 488 Bapta-2 (OGB-2), tetramethylrhodamine ethyl ester (TMRE) perchlorate, benzothiazole coumarin, D-myo-InsP3 P4(5)-1-(2-nitrophenyl)-ethyl ester (caged InsP3), o-nitrophenyl EGTA (NP-EGTA), Mitotracker red, and BODIPY-ryanodine were purchased from Molecular Probes. Dulbeco's Modified Eagles Medium (DMEM) was purchased from GIBCO BRL. All other materials were obtained from Sigma-Aldrich.
Preparation of Pancreatic Acini
Mouse pancreatic acini were prepared essentially as described previously (
Measurement of [Ca2+]i and Flash Photolysis of Caged Compounds
Isolated acinar cells were incubated with 2 µM fura-2 AM at 25°C for 30 min, followed by washing and resuspension in physiological salt solution (PSS) containing (mM): 127 NaCl, 0.56 MgCl2, 4.7 KCl, 0.55 Na2HPO4, 1.28 CaCl2, 10 HEPES-NaOH, 11 D-glucose, pH 7.4. Nominal Ca2+ containing external solution contained the above without Ca2+ and addition of 0.1 mM EGTA. For [Ca2+]i measurement, fura-2loaded cells were placed in a closed recording chamber and mounted on the stage of an Eclipse TE200 microscope (Nikon) equipped with a Nikon Super Fluor 40x, 1.3 NA oil immersion objective. Acini were superfused at 1 ml/min with PSS and rapid solution changes were accomplished by use of a valve attached to a multi-chambered reservoir. Determination of [Ca2+]i was performed using digital imaging microscopy with a monochronometer-based system and high speed CCD camera (T.I.L.L.-Photonics). Cells were alternately excited at 340/380 ± 15 nm and the fluorescence emission collected through a 510 ± 25-nm band pass filter (Chroma). Images were acquired at a rate of 1 Hz. Mean gray values in user-defined areas of interest were used to compute 340/380 ratios. Calibration of fluorescent ratio signals to [Ca2+] was performed using the equation of F/F0, where
F/F0 = 100[(F - F0)/F0], F is the recorded fluorescence, and F0 was obtained from the average of 15 sequential frames after equilibration with the patch pipette solution and before stimulation (
F/F0 images were scaled to 275 levels of gray from a 12-bit scale, between minimum and maximum values. A [Ca2+]i increase was defined as an increase in fluorescence of 25 levels of gray above the minimum scale value. The spread of the wave was determined by measuring the distance between the pixel (pixel size, 0.225 µm) that first increased (corresponding to the trigger zone) and the furthest point at which the [Ca2+]i increased to the previously described threshold value. The wave speed was calculated from the time needed for [Ca2+]i to increase over the specified area and thus represents the mean velocity. Photolytic release of NP-EGTA (caged Ca2+) and caged InsP3 were achieved using a pulsed xenon arc lamp fed to a dual port epifluorescence condenser using a fiber-optic guide (T.I.L.L.-Photonics). An 80-J, 0.5-ms flash of UV light (360 ± 7.5 nm) was reflected onto the plane of focus using a DM400 dichroic mirror and Super Fluor 40x, 1.3 NA oil immersion objective.
Whole-Cell Patch-clamp Recordings
Ca2+-activated Cl- and nonspecific cation currents were recorded at a sampling rate of 1 kHz using an Axopatch 200A patch clamp amplifier (Axon Instruments, Inc.), Instrutech digital interface, and IGOR PRO/Pulse Control XOP software (), cells were superfused with the above solution supplemented with 1% BSA, which was present for patch rupture as well as throughout the experiment. Whole-cell series resistances of 815 M
were achieved after patch rupture. Intervals of 3 min were maintained after patch rupture and between stimuli to allow for equilibration with the patch pipette solution. Cells were maintained at a holding potential of -30 mV. For photolytic release of caged InsP3, the intracellular recording solution contained (mM): 140 KCl, 10 HEPES-NaOH, 1.13 MgCl2, 2 Mg-ATP, 1 n-hydroxyethylethylenediaminetriacetic acid, 0.0010.002 caged InsP3, pH 7.3. For photolytic release of caged Ca2+, the intracellular recording solution contained (mM): 130 KCl, 10 HEPES-NaOH, 10 o-nitrophenyl EGTA, 5 CaCl2, 2 Mg-ATP, 1.2 MgCl2, pH 7.2. Using this intracellular solution, the resting [Ca2+]i and free [Mg2+] were calculated to be 175 nM and 1 mM, respectively.
Determination of the Subcellular Localization of Mitochondria and Ryanodine Receptors
For RyR staining, isolated acinar cells and small clusters were incubated with 0.1 µM BODIPY-ryanodine for 2 h in PSS supplemented with 0.5% BSA. At the end of the incubation, the cells were gently pelleted and washed three times by resuspension in buffer. Localization was examined by laser scanning confocal microscopy using a Noran Oz system. BODIPY-ryanodine was excited at 488 nm and emission was recorded using a 525 ± 25-nm bandpass filter. In some experiments, cells were incubated with 5 µM ryanodine 30 min before incubation with fluorescent probe to determine nonspecific fluorescence. In this case, using identical laser power settings, no measurable signal was detected. For the detection of mitochondrial staining, cells were incubated with 0.1 µM mitotracker red for 2 min, followed by identical wash steps. Mitochondria were visualized by confocal microscopy after excitation of the dye at 565 nm with emission collected above 600 nm.
Estimation of Changes in Mitochondrial Membrane Potential
Isolated acinar cells were loaded with 100 nM TMRE perchlorate for 15 min at room temperature in PSS. 100 nM TMRE was also included in all solutions used for superfusion. Cells loaded with dye were excited at 545 ± 15 nm and fluorescence emission collected using a 565-nm-long pass filter. TMRE distribution was similar to the distribution of fluorescence in cells that were loaded with Mitotracker red. Fluorescence traces were generated from mean gray values using a user-defined region of interest corresponding to regions of high TMRE fluorescence.
Statistical Analysis
For caged Ca2+ experiments, ionic currents were analyzed to determine peak current (Ipeak), total charge (Qtot), and time to steady state recovery (Trec) using IGOR PRO software. Ipeak was defined as the maximum current of the record after photolysis of NP-EGTA and was determined after baseline current subtraction. Qtot was determined by integrating current traces from the time of current activation until recovery of current to baseline or, in some cases, new steady state levels. Trec was defined as the time between Ipeak and recovery to baseline or steady state. Data within groups (control vs. treated) were analyzed using a Wilcoxon test for paired data and between experimental groups (±ryanodine) using a Mann-Whitney test for unpaired, nonparametric data with Graph Pad Prism software. In other experiments, data was analyzed using an appropriate Student's t test. All data is represented as mean ± SEM.
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RESULTS |
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Effect of Mitochondrial Depolarization on Agonist-induced [Ca2+]i Oscillations
Stimulation of acinar cells with low doses of the muscarinic agonist carbachol (CCh) (50250 nM) produced oscillations in [Ca2+]i with a regular frequency (three to six per minute) and amplitude (100300 nM) in nominally Ca2+-free external solution. The oscillations were generally maintained for up to 6 min (
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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-2loaded 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.
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Subcellular Localization of Ryanodine Receptors and Their Role in Agonist-induced [Ca2+]i Signaling
Work by
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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 (
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Activation of CICR through Photolytic Release of Ca2+ after Mitochondrial Depolarization
The aforementioned experiments reveal that Ca2+ release through ryanodine-sensitive stores can be evoked after mitochondrial depolarization, and further suggest that this occurs as a result of colocalization. To determine whether Ca2+ in the vicinity of mitochondria could influence RyR activity, we evoked transient global elevations in [Ca2+]i through photolytic release of caged Ca2+ (
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Physiological Role of CICR from Ryanodine Receptors
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.
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In cells where photolytic release of InsP3 caused Ca2+ release resembling a [Ca2+]i "puff" (i.e., a highly localized, small, and transient response), mitochondrial depolarization had no apparent effect on the propagation of the signal (Fig 7, n = 3). In these experiments, [Ca2+]i remained apically confined, despite mitochondrial depolarization, suggesting that [Ca2+]i increases of this magnitude were not sufficient to recruit participation of mitochondrial Ca2+ import, as has been suggested by
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To investigate whether CICR occurred during normal signaling events, and not solely when mitochondrial Ca2+ import was inhibited, a higher concentration of caged InsP3 (2 µM) was used to photolytically induce global [Ca2+]i increases. This allowed for the direct assessment of the role of RyR on the propagation of global Ca2+ waves. Using this paradigm, global [Ca2+]i increases could be reproducibly evoked (Fig 8D and Fig E). After release of InsP3 under control conditions, 100 µM ryanodine was added to the external bath solution. In the continued presence of ryanodine, sequentially evoked [Ca2+]i responses became progressively more confined to the apical region of the cell compared with control (Fig 8A and Fig B, n = 7). The addition of ryanodine not only prevented the [Ca2+]i increase throughout the basal region, but also caused the signal to retreat well into the apical region, giving increases in a region only slightly more diffuse than the trigger zone. By the fourth uncaging, the [Ca2+]i wave propagated 11.2 ± 1.8 µm (n = 7) in the presence of ryanodine, compared with 15.7 ± 1.3 µm in time-matched controls (Fig 8 C, n = 10, P = 0.006). This decrease in propagation represented a reduction in the distance traveled across the cell from 95.5 ± 2.9% of the diameter of the cell to 63.9 + 10.9% in the presence of ryanodine. In addition, the rate of propagation was significantly slowed in the presence of ryanodine, traveling at 21.2 ± 6 µm/s (n = 10) in the absence of and 9.5 ± 2.3 µm/s (n = 7) after incubation in ryanodine for 12 min (P = 0.03). The latency after the flash to the initiation of the signal was not significantly altered by treatment with ryanodine (192 ± 5 vs. 175 ± 2 ms, control vs. treated after 12 min, n = 4), as expected for a signal initiated through InsP3R. Interestingly, the spatially limiting effects of RyR blockade could be overcome at higher agonist concentrations (Fig 8A V and B V).
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DISCUSSION |
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In pancreatic acinar cells, Ca2+ signaling is initiated by the binding of InsP3 to its receptor on specialized portions of the ER present in the extreme apical regions of the cell, the so-called trigger-zone (
Numerous studies in a wide variety of cell types have demonstrated that mitochondria play an important role in buffering changes in [Ca2+]i under physiological as well as pathological situations (for review, see
Several pieces of experimental evidence indicate that the activity of the RyR is influenced by the ability of mitochondria to buffer [Ca2+]i in their immediate vicinity. First, during stimulation with physiological concentrations of agonist, mitochondrial depolarization results in a large [Ca2+]i increase. However, this [Ca2+]i increase may not simply be the result of decreased mitochondrial buffering and release of stored mitochondrial Ca2+. Since the [Ca2+]i increase was largely blocked by either emptying ER Ca2+ stores or by inhibition of RyR, this initial observation may indicate that Ca2+ release from the ER is the important event after limited release of mitochondrial Ca2+ as the initial trigger. The attenuation of the response by ryanodine is indicative of a CICR event mediated by RyR. An alternative explanation is that, under conditions of physiological stimulation, mitochondria preferentially import Ca2+ released by RyR. Thus, treatment with ryanodine would be expected to lead to decreased mitochondrial sequestration of Ca2+ and the resultant refractory response to FCCP. This interpretation would also support the notion of a microdomain of mitochondria and ER expressing RyR and is consistent with the view that mitochondria preferentially sense rapid, oscillatory changes in [Ca2+]i (
Mitochondrial depolarization after photolytically induced global [Ca2+]i increases resulted in a significant augmentation of [Ca2+]i as evidenced by the delayed recovery of Ca2+-activated currents in the presence of FCCP. Similar effects have been observed in sympathetic neurons after depolarization-induced Ca2+ influx (
Uncaging threshold concentrations of InsP3 induced apically localized Ca2+ signals; a Ca2+ wave was generated that was restricted to the zymogen granule-containing region and did not invade the basal region of the cell. Mitochondrial depolarization resulted in the transition from a spatially limited response to a global Ca2+ signal. The global increase was largely attenuated by treatment with ryanodine, suggesting that the transition from a local to global Ca2+ signal following mitochodrial depolarization was dependent on a CICR event. It also follows that the signal is normally limited in the presence of mitochondria and as a result of buffered or limited RyR activation. In some experiments, the smallest and most transient signals were unaffected by mitochondrial depolarization. These data would indicate that the smallest signals appear limited by processes such as clearance of Ca2+ by plasma membrane and ER ATPases, cytoplasmic buffering, as well as the metabolism of InsP3, and not by mitochondrial buffering.
Data showing that inhibitory concentrations of ryanodine result in a global [Ca2+]i signal being spatially limited to the apical region of the cell is strong evidence that RyR play an important role in wave propagation out of the trigger zone under physiological conditions, and not simply when mitochondrial import is compromised. The observation that the wave speed of the residual apical signal was slowed significantly in the presence of ryanodine also indicates that CICR plays a role in propagating the signal from the initial InsP3-induced release. This is consistent with the reduction in wave speed noted in the presence of ryanodine by
Ca2+ signals are apparently capable of propagating across cells in the absence of CICR through RyR, as indicated by the observation that high concentrations of agonist were able to overcome inhibition by ryanodine, resulting in an apical-to-basal global Ca2+ wave. These data are consistent with the demonstration that low levels of InsP3R are expressed throughout the cytoplasm of acinar cells (
In conclusion, the present study indicates a continuum of events that determine whether a [Ca2+]i signal is localized to the apical region or spreads as a wave throughout the cell. The initial common trigger appears to be InsP3-induced Ca2+ release from the apical trigger zone. At all but the lowest concentrations of agonist, apically localized signals are confined to this region by the buffering capacity of mitochondria. At greater stimulus strength, the buffering capacity of mitochondria is overcome as CICR through RyR becomes the dominant mechanism for propagating a global Ca2+ wave.
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Footnotes |
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Drs. Straub and Giovannucci contributed equally to this work and should be considered co-first authors.
1 Abbreviations used in this paper: caged-InsP3, D-myo-InsP3 P4(5)-1-(2-nitrophenyl)-ethyl ester; CCh, carbamylcholine; CICR, Ca2+-inducedCa2+ release; ER, endoplasmic reticulum; InsP3, inositol 1,4,5-trisphosphate; NP-EGTA, o-nitrophenyl ethylenediaminetriacetic; OGB-2 Oregon green 488 Bapta-2; PSS, physiological salt solution; RyR, ryanodine receptor; TMRE, tetramethylrhodamine ethyl ester.
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Acknowledgements |
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The authors thank Drs. T. Shuttleworth, S.S. Sheu, P. Hinkle, and E. Stuenkel for helpful comments throughout the study and during preparation of the manuscript.
This study was supported by the National Institute of Diabetes and Digestive and Kidney Diseases grant DK-54568 (D.I. Yule).
Submitted: 28 June 2000
Revised: 21 August 2000
Accepted: 21 August 2000
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