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Endocrinology Vol. 144, No. 8 3565-3574
Copyright © 2003 by The Endocrine Society

Ca2+-Induced Ca2+ Release in the Pancreatic ß-Cell: Direct Evidence of Endoplasmic Reticulum Ca2+ Release

Thomas K. Graves and Patricia M. Hinkle

Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York 14642

Address all correspondence and requests for reprints to: Patricia M. Hinkle, Ph.D., University of Rochester Medical Center, Box 711, 601 Elmwood Avenue, Rochester, New York 14642. E-mail: patricia_hinkle{at}urmc.rochester.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The role of the Ca2+-induced Ca2+ release channel (ryanodine receptor) in MIN6 pancreatic ß-cells was investigated. An endoplasmic reticulum (ER)-targeted "cameleon" was used to report lumenal free Ca2+. Depolarization of MIN6 cells with KCl led to release of Ca2+ from the ER. This ER Ca2+ release was mimicked by treatment with the ryanodine receptor agonists caffeine and 4-chloro-m-cresol, reversed by voltage-gated Ca2+ channel antagonists and blocked by treatment with antagonistic concentrations of ryanodine. The depolarization-induced rise in cytoplasmic Ca2+ was also inhibited by ryanodine, which did not alter voltage-gated Ca2+ channel activation. Both ER and cytoplasmic Ca2+ changes induced by depolarization occurred in a dose-dependent manner. Glucose caused a delayed rise in cytoplasmic Ca2+ but no detectable change in ER Ca2+. Carbamyl choline caused ER Ca2+ release, a response that was not altered by ryanodine. Taken together, these results provide strong evidence that Ca2+-induced Ca2+ release augments cytoplasmic Ca2+ signals in pancreatic ß-cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INTRACELLULAR CA2+ SIGNALS play a pivotal role in the function of the pancreatic ß-cell (1). This cell’s most important role is the secretion of insulin, and knowledge of the intricacies of the Ca2+ signals involved in excitation-secretion coupling is important in understanding both normal ß-cell function and pathological states. Control of the concentration of cytoplasmic Ca2+ ([Ca2+]c) in ß-cells is complex. Influx of Ca2+ through L-type voltage-dependent Ca2+ channels (VDCC) in response to depolarization is a well-established mechanism of [Ca2+]c increase in response to high extracellular glucose (2, 3, 4). Endoplasmic reticulum (ER) Ca2+ stores are filled from the cytoplasm by the sarcoplasmic/ER Ca2+ ATPase (SERCA) (5). Efflux across the plasma membrane (6) and mitochondrial Ca2+ buffering (7, 8) also regulate fluctuations in [Ca2+]c. ER Ca2+ stores can be mobilized through generation of 1,4,5-inositol trisphosphate (IP3) in response to activation of receptors coupled to phospholipase C, and subsequent gating of the IP3 receptor Ca2+ channel on the ER membrane (9, 10). Dense core secretory vesicles also form a significant and highly regulated Ca2+ store in pancreatic ß-cells (11, 12).

In addition to these pathways, Ca2+ from ryanodine-sensitive stores may contribute to changes in [Ca2+]c. The ryanodine receptor (RyR) is an ER Ca2+ channel that is gated by Ca2+ itself (13) and releases ER Ca2+ in response to increases in [Ca2+]c, a process called Ca2+-induced Ca2+ release (CICR). This mechanism of Ca2+ signal augmentation is well established in myocytes (14, 15, 16), but the function of RyRs in the ß-cell is less well understood. Pancreatic ß-cells and various ß-cell lines have been reported to express RyRs and display CICR (17, 18, 19, 20, 21, 22, 23, 24). Lemmens et al. (25) showed recently that depletion of [Ca2+]er (concentration of ER Ca2+) with thapsigargin, a SERCA inhibitor, abolished a second phase of [Ca2+]c increase induced by KCl depolarization of islet cells from ob/ob mice, suggesting that CICR from the ER may contribute to the [Ca2+]c signal generated by L-type VDCC activation. A number of studies have shown release of ER Ca2+ via RyRs in response to the RyR agonists caffeine and 4-chloro-3-ethylphenol (8, 26), implying that ß-cells have functional RyRs. Glucagon-like peptide-1 (GLP-1), an insulin secretagogue that increases cAMP, stimulates CICR, in part, by a ryanodine-sensitive pathway (8). This pathway involves the cAMP-regulated exchange factor Epac and is coupled to exocytosis (8, 20, 27, 28).

In contrast, two independent groups recently reported that insulin secretagogues that increase [Ca2+]c cause corresponding elevations [Ca2+]er (21, 29). The most marked increases in [Ca2+]er occur when [Ca2+]c is transiently raised to the highest concentration by K+ depolarization. Furthermore, a study using ER-targeted "cameleon" (30) Ca2+ indicators expressed in insulin-secreting MIN6 cells (a mouse insulinoma line) showed that depolarizing concentrations of KCl and nutrient insulin secretagogues lead to an apparent increase in [Ca2+]er (8). These findings would seem to belie a CICR mechanism in ß-cells, because the model would predict that sharp increases in [Ca2+]c would activate the RyR Ca2+ channel and reduce [Ca2+]er. Uptake and metabolism of glucose leads to an increase in intracellular ATP (3, 31), which could enhance SERCA activity to a point where ER Ca2+ influx would overshadow efflux of Ca2+ through the RyR. In addition, the experimental methods involved in some prior studies of depolarization- and glucose-induced modulation of [Ca2+]er involved permeabilizing (29) and Ca2+-starving (21) cells, manipulations that may have altered the normal ER Ca2+ homeostasis.

We report here the results of experiments using ER cameleon Ca2+ indicators, as well as cytoplasmic Ca2+-indicating dyes, to study CICR in intact MIN6 cells maintained in normal extracellular Ca2+. MIN6 cells were chosen because they are well differentiated and exhibit mechanisms of control of insulin transcription and secretion similar to those governing normal ß-cells (32) and because they are relatively easy to culture, transfect, and study by fluorescence microscopy. We provide direct evidence that CICR contributes to Ca2+ responses in MIN6 cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Ryanodine and fura2/AM were purchased from Molecular Probes (Eugene, OR). Fura2FF/AM, 1,2-bis(2-aminophenoxy)ethane-N, N, N', N'-tetraacetic acid (BAPTA), and thapsigargin were obtained from Calbiochem (La Jolla, CA). Lipofectamine reagent was from GIBCO (Grand Island, NY), and 4-chloro-m-cresol was purchased from Fluka (Buchs, Switzerland). DMEM was purchased from Sigma (St. Louis, MO). Cellgro heat-inactivated fetal calf serum was purchased from Mediatech (Herndon, VA). MIN6 cells were generously provided by Dr. J. Miyazaki (Osaka University, Osaka, Japan), and INS-1 cells by Dr. C. Wollheim (University of Geneva, Geneva, Switzerland). The plasmid encoding the cameleon ER Ca2+ indicator, YC4er, was a gift from Dr. R. Tsien (University of California-San Diego). BAY K8644 was from RBI (Natick, MA), and [45Ca]CaCl2 and [3H]myo-inositol from Dupont/NEN Life Science Products (Boston, MA).

Cell culture
MIN6 cells were grown in DMEM (25 mM glucose) supplemented with 15% fetal calf serum; passages 16–25 were used in all experiments. For immunocytochemistry and for calcium imaging studies, cells were seeded on 25-mm glass coverslips in 35-mm plastic dishes and grown to approximately 70% confluence. Each dish contained approximately 700 µg protein.

Single-cell cytoplasmic calcium imaging
Twenty-four hours before imaging, culture medium was replaced with medium containing 3 mM glucose. In preparation for imaging, culture medium was removed, and cells were incubated in Krebs buffered ringers solution (KBRS) [117 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 5 mM NaHCO3, 20 mM HEPES, 0.1% BSA, and 3 mM glucose (pH 7.4)] containing 4 µM fura2/AM or fura2FF/AM and 1 µg/ml cyclosporin at room temperature for 45 min. Cyclosporin was included in the loading reaction because it reduces efflux of the esterified forms of the indicators and increases loading (33); cyclosporin was not present during experimental incubations, and its presence during loading did not affect results. Fura2FF [dissociation constant (Kd), 12 µM) (34) was used to study depolarization-induced increases in [Ca2+]c, which were too large to be reported accurately by the higher-affinity dye fura2 (Kd, 190 nM) (35). Coverslips were rinsed in KBRS and placed in a Sykes-Moore chamber (Bellco Glass, Vineland, NJ), on a 37 C heated stage of a Nikon Diaphot inverted microscope fitted with a 150-W xenon lamp, and allowed to equilibrate for 20 min. Additions were made by removing 250 µl of the buffer bathing the cells, mixing it with any drug, and adding the solution back to the chamber over a period of 2 sec or less. Ratios of 510 nm emission at 340 vs. 380 nm excitation wavelengths were acquired every 3 sec, using a Princeton Instruments Micromax (Princeton, NJ) camera. Filters were changed using a {lambda} 10–2 optical filter changer from Sutter Instrument (Novato, CA). The filter changer and camera were controlled, and images of 15–25 cells from each field were analyzed using MetaFluor software from Universal Imaging (Downingtown, PA). To facilitate comparison between experiments, data are expressed as 340/380-nm ratios normalized to the baseline ratio.

ER calcium imaging
MIN6 cells, growing on glass coverslips in 35-mm dishes, were transfected with 1 µg of the plasmid encoding YC4er in 1 ml of serum-free DMEM with 15 µl Lipofectamine Reagent. After a 4-h incubation, the reaction mixture was replaced with DMEM containing serum, and the cells were grown for 2–4 d. Twenty-four hours before the start of experiments, cells were placed in medium containing 3 mM glucose. Coverslips were washed with KBRS and placed in a Sykes-Moore chamber and allowed to equilibrate for 20 min at 37 C. Cells expressing YC4er were recognized by the reticular pattern of fluorescence seen throughout the cytoplasm, and 5–10 cells from each field were selected for data collection. An excitation wavelength of 440 nm was used, and emission acquisitions at 535 and 485 nm were made every 10 sec, using Metafluor software. The long interval between acquisitions was necessary to prevent excessive photobleaching. Data are presented as changes in the 535/485-nm emission ratio relative to the starting ratio. Because the YC4er reporter was not saturated with Ca2+ under any conditions tested, it was not feasible to convert 480/535 fluorescence emission ratios to reliable [Ca2+]er values.

45Ca2+ Influx Studies
Cells on 35-mm dishes were incubated in KBRS exactly as described for calcium imaging studies except that the total calcium concentration was reduced to 0.2 mM. Cells were preincubated for 45 min in buffer with or without 100 µM ryanodine. Then, [45Ca]CaCl2 (2 µCi/ml) was added with or without 1 µM BAY K8644 and 25 mM KCl, and incubation was continued for 5 min at 37 C. Dishes were washed three times with 2 ml ice-cold saline and solubilized in 0.1% sodium dodecyl sulfate; radioactivity was quantified in a liquid scintillation counter.

Inositol phosphate studies
Cells on 35-mm dishes were incubated overnight in medium containing dialyzed fetal bovine serum and 2.5 µCi/ml [3H]myo-inositol. Dishes were washed with KBRS and then incubated for 40 min with KBRS containing 10 mM LiCl and other additions as noted. [3H]Inositol phosphates were isolated by ion-exchange chromatography as previously described (36).

Immunocytochemistry
Immunocytochemistry procedures were performed at room temperature. MIN6 cells on glass coverslips were fixed in 4% paraformaldehyde for 5 min and then washed three times with PBS. The cells were permeabilized and blocked by incubating in PBS containing 0.2% Nonidet P-40 and 5% goat serum for an additional 20 min. Cells were then incubated in the same blocking buffer containing 1:1000 dilutions of rabbit anticalreticulin 405–417 from Calbiochem or mouse anti-green fluorescent protein (anti-GFP) from Roche Molecular Biochemicals (Indianapolis, IN), for 60 min, followed by four 5-min washes in PBS to remove unbound primary antibody. Coverslips were then incubated for 20 min in blocking buffer containing 1:200 dilutions of fluorescein-isothiocyanate-labeled goat-antimouse IgG and rhodamine-labeled goat antirabbit IgG from Molecular Probes. Coverslips were mounted on glass slides in mowiol supplemented with 2.5% 1,4-diazabicyclo-[2,2,2]-octane, and images were obtained on a Leica TCSSP confocal microscope (Leica Microsystems, Chantilly, VA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of YC4er in MIN6 cells
The cameleon YC4er was used to detect changes in [Ca2+]er (30, 37, 38). In brief, YC4er, which binds Ca2+ with Kd values of 700 µM and 80 nM, is a construct made of two variants of GFP linked by calmodulin and a Ca2+-calmodulin binding domain. The lower-affinity binding site is suitable for observing changes in [Ca2+]er, which is estimated to be 250 µM in resting MIN6 cells (8). When Ca2+ binds to calmodulin, the interaction with the Ca2+-calmodulin binding domain causes a conformational change, resulting in an increase in fluorescence resonance energy transfer. Changes in fluorescence resonance energy transfer are detected by measuring the 535/485-nm emission ratio after excitation at 440 nm. Increases or decreases in the 535/485-nm ratio reflect the same directional changes in [Ca2+]er.

Using the plasmid encoding YC4er, we achieved transfection rates of approximately 5% in most experiments. Confocal microscopy revealed that the cameleon, visualized with antibody against GFP, colocalized with the ER marker calreticulin (Fig. 1Go). The cameleon was excluded from the nucleus and was not concentrated in the Golgi apparatus or visible on the plasma membrane.



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  		FIG. 1. Localization of YC4er cameleon in MIN6 cells. Cells expressing YC4er were fixed and then incubated with antibody against either GFP (to localize the YC4er cameleon) or calreticulin (an ER marker). Confocal images through the center of the cell are shown. The cameleon is shown in green, calreticulin in red, and a merged image in the right panel, where orange and yellow represent regions of colocalization. Colocalization of the two proteins seems to be essentially complete. Images obtained on a standard fluorescence microscope show more clearly that cameleon is not visible in the Golgi apparatus, which is relatively large in MIN6 cells. Control experiments showed that there was no significant background when primary antibodies were omitted. Most cells showed no GFP staining in transfected cultures, reflecting the low transfection efficiency, and no GFP staining was observed in untransfected control cultures.

 
Depolarization-induced changes in [Ca2+]er
In myocytes, rapid influx of Ca2+ through VDCCs results in CICR from the sarcoplasmic reticulum through the RyR. This is a central mechanism of excitation-contraction coupling in muscle cells. Based on evidence supporting the presence of a RyR in pancreatic ß-cells, we asked whether a similar mechanism of depolarization-induced CICR exists in excitable MIN6 cells. Depolarizing concentrations of KCl result in marked, rapid, and sustained elevations in [Ca2+]c in these cells, and we hypothesized that depolarization would cause an observable release of Ca2+ from the ER.

Depolarization of the plasma membrane in MIN6 cells expressing ER-targeted cameleons caused a release of ER Ca2+, as indicated by a decrease in the 535/485-nm ratio (Fig. 2AGo). KCl was added isotonically in all experiments. The decrease in [Ca2+]er began within 20 sec of depolarization, when the first point was obtained, and the 535/485 ratio decreased to its minimal level with addition of a mixture of thapsigargin (to block SERCA activity), BAPTA (to chelate extracellular Ca2+), and ionomycin (an ionophore which allows efflux of Ca2+ from the ER) (Fig. 2AGo). The vehicle control showed no drop in [Ca2+]er, but slow photobleaching was seen (Fig. 2EGo). When the SERCA inhibitor cyclopiazonic acid (CPA) and BAPTA were added to block ER refilling, [Ca2+]er declined as the ER pool became depleted, and there was no additional response to ionomycin (Iono) (Fig. 2FGo).



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  		FIG. 2. Dynamic changes in [Ca2+]er. MIN6 cells transfected with YC4er were incubated in KBRS and treated as indicated. A, KCl (25 mM) was added isotonically as indicated by the line. B, Diltiazem (20 µM) was added before depolarization with 25 mM KCl. C, Caffeine (20 mM), a RyR agonist, was added as indicated. D, Cells were incubated with 100 µM ryanodine for 45 min before the start of imaging, and ryanodine was present throughout the experiment; 25 mM KCl was added as indicated. E, Vehicle (KBRS) was added as indicated. Each trace represents mean ± SEM of 6–10 single cell recordings. F, Solid line, CPA (50 µM) and 3 mM BAPTA were added together, and 500 nM ionomycin was added at 350 sec, as shown; broken line, CPA, BAPTA, and ionomycin were added simultaneously. This experiment was done at a different time from those shown in A–E, accounting for the difference in absolute ratio values. All experiments were repeated at least three times, with similar results. TBI indicates addition of 1 µM thapsigargin, 3 mM BAPTA, and 500 nM ionomycin to cause maximal drops in [Ca2+]er. Data are expressed as baseline-normalized 535/485-nm ratios.

 
The KCl-induced decrease in [Ca2+]er was dose dependent (Fig. 3BGo), and the maximum change was observed with 25 mM KCl. We used fura2FF to detect rises in [Ca2+]c on depolarization with KCl and found a similar dose dependence, with a maximum rise in [Ca2+]c at 20 mM KCl (Fig. 3AGo). These dose-response relationships are not directly comparable, however, because the peak in [Ca2+]er occurred earlier than the maximal depletion of the ER Ca2+ pool.



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  		FIG. 3. Effects of KCl concentration on the peak height of [Ca2+]c and on release of ER Ca2+. MIN6 cells were incubated in KBRS and depolarized with increasing concentrations of KCl. The upper panel shows fura2FF-indicated peaks from 25 single cells (mean ± SEM) for each KCl concentration tested. In the bottom panel, the decrease in 535/485-nm ratio for each KCl concentration is expressed as a percentage of the maximal decrease obtained after ER Ca2+ pools were depleted with 1 µM thapsigargin, 3 mM BAPTA, and 500 nM ionomycin. Each bar represents 6–10 YC4er-transfected MIN6 cells.

 
The KCl-induced decline in the 535/485-nm ratio was prevented by 20 µM diltiazem, an L-type VDCC blocker (Fig. 2BGo), suggesting that the decreases in [Ca2+]er shown in Figs. 2AGo and 3AGo were attributable to influx of Ca2+ through VDCCs. When added after KCl, diltiazem caused a prompt fall in [Ca2+]c and a gradual increase in [Ca2+]er as ER Ca2+ stores refilled (Fig. 4Go). The KCl-induced fall in [Ca2+]er was also partially reversed by representatives of two other distinct classes of VDCC antagonists, nifedipine and methoxy-verapamil (data not shown). These findings all point to the conclusion that discharge of ER Ca2+ stores depends on Ca2+ influx through VDCCs.



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  		FIG. 4. Reversibility of KCl-induced changes in [Ca2+]c and [Ca2+]er. MIN6 cells were loaded with fura2 to detect changes in [Ca2+]c (A) or transfected with YC4er to detect changes in [Ca2+]er (B). KCl (25 mM) and 20 µM diltiazem were added as shown by the bars. Traces represent mean ± SEM from 15 (A) or 6 (B) cells.

 
The KCl-induced decrease in [Ca2+]er was mimicked by treatment with 20 mM caffeine (Fig. 2CGo), a RyR agonist, and was blocked almost completely by 100 µM ryanodine (Fig. 2DGo).

In the studies described, cells were grown in high glucose (25 mM) but were switched to 3 mM glucose the night before imaging studies and maintained in 3 mM glucose during experiments unless noted. The ability of KCl to discharge ER Ca2+ was not different if the cells were incubated in 0 mM glucose.

Based on the finding of ER Ca2+ release in response to KCl, we expected CICR to contribute to the increase in [Ca2+]c on depolarization with KCl. To test this prediction, we compared fura2FF-indicated rises in [Ca2+]c induced by 25 mM KCl in cells treated with ryanodine or with vehicle. Ryanodine blunted the rise in [Ca2+]c caused by depolarization with 25 mM KCl (Fig. 5AGo). We tested whether depletion of ER Ca2+ stores with the irreversible SERCA inhibitor thapsigargin could eliminate the ryanodine-sensitive portion of the KCl-induced rise in [Ca2+]c (Fig. 5BGo). After treatment with thapsigargin, there was no difference in the KCl-induced rise in [Ca2+]c in cells treated with ryanodine vs. vehicle. Because ER Ca2+ stores were depleted, this result also shows that ryanodine itself did not directly affect Ca2+ influx through VDCCs. The fact that [Ca2+]c responses to depolarization were not diminished in cells that had been treated with thapsigargin, when CICR had been lost, implies that store depletion stimulated an influx pathway.



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  		FIG. 5. Effect of ryanodine on KCl-induced rise in [Ca2+]c with or without thapsigargin treatment. A, Single-cell fura2FF-indicated [Ca2+]c responses are shown in MIN 6 cells that had been preincubated for 45-min pretreatment without (fine upper trace) or with 100 µM ryanodine (bold lower trace). B, MIN6 cells were incubated for 30 min with 1 µM thapsigargin to deplete ER Ca2+ stores and incubated without (fine trace) or with 100 µM ryanodine (bold trace) as above. Ryanodine, but not thapsigargin, was present during imaging. In both experiments, 25 mM KCl and 3 mM BAPTA were added as indicated. Each trace represents baseline-normalized 340/380-nm ratios from 15–25 cells (mean ± SEM). In A, 340/380 ratios averaged 0.265 ± 0.005 and 0.277 ± 0.008 for untreated and ryanodine-treated cells, respectively. In B, baseline 340/380 ratios were 0.28 ± 0.01 under both conditions. Control experiments confirmed that thapsigargin caused the expected increase in [Ca2+]c, but [Ca2+]c recovered to basal levels within 45 min. This explains why the 340/380-nm ratios were essentially the same at the start of imaging regardless of thapsigargin pretreatment.

 
The 100-µM concentration of ryanodine tested here is the concentration used typically in studies of excitation:contraction coupling, and it has been shown that ryanodine does not inhibit VDCC activity in cardiac and skeletal muscle (39, 40, 41). Ryanodine also inhibited the KCl-evoked discharge of ER Ca2+ at 10 µM (Fig. 6Go). To rule out the possibility that ryanodine inhibited VDCC in our experiments, we quantified 45Ca2+ uptake into ryanodine- or vehicle-treated MIN6 cells in response to 25 mM KCl and the L-channel agonist BAY K8644. At concentrations up to 100 µM, ryanodine had no effect on basal or depolarization-stimulated 45Ca2+ influx, indicating that VDCC activation was not altered (Fig. 7Go).



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  		FIG. 6. Effect of 10 µM ryanodine on KCl-induced decrease in [Ca2+]er. MIN6 cells transfected with YC4er were pretreated as follows: A, preincubation for 45 min with vehicle [DMSO (dimethylsulfoxide)]; B, preincubation for 45 min with 10 µM ryanodine. Cells were then incubated in KBRS containing ryanodine, at the same concentrations as those used for pretreatment, and exposed to 25 mM KCl and 1 µM thapsigargin, 3 mM BAPTA, and 500 nM ionomycin (TBI) as indicated on the figure. Each trace represents the mean ± SEM of 6–10 single cell recordings. Data are expressed as baseline-normalized 535/485-nm ratios.

 


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  		FIG. 7. Effect of ryanodine on 45Ca2+ uptake in MIN6 cells. Cells were incubated with or without 100 µM ryanodine for 45 min and then incubated for 5 min in KBRS with 0.2 mM CaCl2 containing 45Ca2+ and either no stimulus (light bars) or 25 mM KCl and 1 µM BAY K8644 (dark bars). Values shown are the mean and range of duplicate determinations.

 
4-Chloro-m-cresol responses
When MIN6 cells were treated with 500 nM 4-chloro-m-cresol, a drug that activates the RyR (17, 42), there was a marked increase in [Ca2+]c, and this increase was inhibited almost completely by pretreatment with ryanodine (Fig. 8Go). These results provide additional evidence that Ca2+ can be released from the ER of MIN6 cells through the RyR. After exposure to 500 nM 4-chloro-m-cresol for 2–3 min, ionomycin-releasable ER Ca2+ pools were diminished by an average of 90% (data not shown). When extracellular Ca2+ was removed to prevent uptake of extracellular Ca2+, with or without nimodipine, the peak [Ca2+]c responses to 4-chloro-m-cresol were decreased by an average of 54%, whereas total ER pool sizes, estimated from the release with ionomycin, were reduced by 70%. Although the intensity of the response to 4-chloro-m-cresol varied between experiments, these results show that the major effect of the drug was to release Ca2+ from intracellular pools.



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  		FIG. 8. 4-Chloro-m-cresol responses in MIN6 cells. MIN6 cells were preincubated without (fine trace) or with 100 µM ryanodine, which was present throughout the experiment (bold trace), and loaded with fura2FF; 500 nM 4-chloro-m-cresol and 3 mM BAPTA were added as shown. Data are expressed as baseline-normalized 340/380-nm ratios. Baseline 340/380-nm ratios were 0.215 ± 0.005 for both traces. Traces represent mean (± SEM) of 15–20 individual cells.

 
Phospholipase C responses
Because phospholipase C activity is increased by Ca2+, some of the KCl-mediated discharge of ER Ca2+ could have resulted from formation of IP3 and activation of the IP3 receptor. To estimate the importance of this pathway, we measured [3H]inositol phosphates in metabolically labeled cells exposed to either 25 mM KCl or an agonist that activates phospholipase C via a G-protein-coupled receptor, carbamyl choline (CCh). As shown in Fig. 9Go, depolarization caused only a slight (60%) increase in total inositol phosphates, whereas the muscarinic receptor agonist caused an 18-fold increase. These findings indicate that an increase in [Ca2+]c by itself causes little increase in IP3 and suggests that efflux via the IP3 receptor is unlikely to contribute much to the decrease in [Ca2+]er caused by depolarization. This conclusion is consistent with the ability of ryanodine to block the loss of ER Ca2+ caused by depolarization, but not CCh, as shown below.



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  		FIG. 9. Rate of inositol phosphate formation in MIN6 cells. Cells were metabolically labeled with [3H]myo-inositol and then incubated in KBRS containing 10 mM LiCl and no additions, 1 mM carbamyl choline, or 25 mM KCl. Total [3H]-inositol phosphates were then isolated. Bars show the mean ± SEM of triplicate dishes.

 
Glucose-induced changes in [Ca2+]c and [Ca2+]er
Because pancreatic ß-cells act as glucose sensors and secrete insulin in response to increases in extracellular glucose concentrations, we studied the effects of glucose on [Ca2+]c and [Ca2+]er to determine whether CICR plays a role in control of the Ca2+ signal induced by glucose. Metabolism of glucose in normal ß-cells results in an increase in the ratio of ATP to ADP, inhibition of the KATP channel, and subsequent depolarization of the cell membrane, resulting in influx of Ca2+ through VDCCs (3, 31). We observed increases in [Ca2+]c in response to 30 mM glucose in 87% of MIN6 cells, using fura2 as the indicator, but the increase was not detectable with the lower affinity reporter fura2FF. A typical glucose response, which occurred with a lag of about 2 min, is depicted in Fig. 10AGo. The rise in [Ca2+]c in response to glucose was much smaller in magnitude, and more gradual than that induced by 25 mM KCl. Furthermore, this glucose-induced increase in [Ca2+]c was not accompanied by a corresponding change in [Ca2+]er (Fig. 10BGo). Because agents that increase cAMP intensify the ß-cell’s response to glucose, we tested the effect of two agents that activate adenylyl cyclase, the peptide GLP-1 and forskolin. Both GLP-1 and forskolin caused a marked increase in the frequency and amplitude of cytoplasmic Ca2+ transients but did not alter [Ca2+]er with or without high glucose, and ryanodine had no effect in these experiments (data not shown). Ryanodine did, however, cause a delay in the peak in [Ca2+]c induced by glucose, although the height of the glucose-induced [Ca2+]c peak was similar with or without ryanodine treatment (Fig. 11Go). After the glucose-induced rise in [Ca2+]c the cells were treated with ionomycin, in the presence of extracellular BAPTA, to allow release of Ca2+ from the ER. The peak in [Ca2+]c after ionomycin treatment was higher in ryanodine-treated than in vehicle-treated cells, but the relative rise in [Ca2+]c (from baseline to peak) was not different between ryanodine- and vehicle-treated cells. The finding that ryanodine altered the kinetics of the cytoplasmic Ca2+response to glucose, but did not have an observable effect on ER Ca2+, probably reflects the low sensitivity of the ER Ca2+ indicator but could also point to actions of ryanodine at sites other than RyRs. Addition of 30 mM mannitol did not affect [Ca2+]c or [Ca2+]er.



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  		FIG. 10. Effects of glucose on [Ca2+]c and [Ca2+]er in MIN6 cells. Cells were maintained in 3 mM glucose before experiments. A, Representative trace showing fura2-indicated [Ca2+]c in 15–25 single cells (mean ± SEM). Cells were loaded with fura2 and then treated with 30 mM glucose as indicated. B, Representative trace of YC4er-indicated [Ca2+]er in 6–10 cells in response to 30 mM glucose. Cells were transfected with YC4er 2–4 d before imaging. Lines labeled TBI indicate addition of 1 µM thapsigargin, 3 mM BAPTA, and 500 nM ionomycin to cause maximal drops in [Ca2+]er.

 


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  		FIG. 11. Effects of ryanodine on glucose-induced rise in [Ca2+]c in MIN6 cells. MIN6 cells were incubated in medium containing 3 mM glucose for 24 h and then loaded with fura2 and treated with 30 mM glucose at 30 sec as shown. Cells were pretreated for 45 min with vehicle (fine trace) or 100 µM ryanodine, which remained throughout the experiment (bold trace). Glucose (30 mM), 3 mM BAPTA, and 500 nM ionomycin were added as indicated. Traces represent mean [Ca2+]c responses from 15–25 individual cells; for clarity, only a few representative error bars are shown.

 
Because glucose caused a slow increase in [Ca2+]c, whereas depolarization with high potassium caused a rapid rise, we asked whether the rate of increase in [Ca2+]c affected [Ca2+]er. To do so, we varied the rate of depolarization (and hence, Ca2+ influx) and measured [Ca2+]er (Fig. 12Go). When 25 mM KCl was added rapidly, over 1–2 sec, it caused a rapid influx of Ca2+ and induced CICR. Gradual addition of 25 mM KCl over a 2-min period, however, promoted a more gradual increase in [Ca2+]c and resulted in very little, if any, CICR. These results suggest that the failure to observe CICR in response to high glucose may be partially attributable to the slow time course of the glucose-induced [Ca2+]c rise.



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  		FIG. 12. Effect of rate of depolarization on CICR in MIN6 cells. Upper panel, KCl (25 mM) was added, over 1–2 sec, at the time indicated. Lower panel, KCl was increased by 2.5 mM, every 12 sec, over a 2-min period between 30 and 150 sec, to give a final concentration of 25 mM. Each trace represents YC4er-indicated [Ca2+]er (mean ± SEM) from 6–10 cells. TBI indicates addition of 1 µM thapsigargin, 3 mM BAPTA, and 500 nM ionomycin. This experiment was repeated three times, with similar results.

 
CCh-induced changes in [Ca2+]c and [Ca2+]er
CCh acts to raise [Ca2+]c in the pancreatic ß-cell by stimulating phospholipase C-coupled muscarinic acetylcholine receptors, resulting in generation of IP3. When MIN6 cells were exposed to 100 µM CCh, [Ca2+]er decreased (Fig. 13AGo). There was no significant difference in the CCh-induced decline in [Ca2+]er in ryanodine-treated vs. vehicle-treated cells. When fura2-loaded MIN6 cells were exposed to 100 µM CCh, [Ca2+]c increased rapidly (Fig. 13BGo), but the increase in [Ca2+]c was small relative to that seen with depolarization. The increase in [Ca2+]c was similar in cells treated with ryanodine or with vehicle (Fig. 13BGo); although, in some experiments, the Ca2+ transient returned to baseline more rapidly in ryanodine-treated cells. CCh did not deplete the ER Ca2+ pool completely, because ionomycin addition led to a further decrease in [Ca2+]er and increase in [Ca2+]c (data not shown). A direct comparison between the responses to CCh and KCl, indicated by fura2, is shown in Fig. 13CGo; fura2FF did not reproducibly report a change with CCh.



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  		FIG. 13. Effects of ryanodine on CCh response in MIN6 cells. In both panels, responses from cells pretreated with vehicle are represented by fine traces, and cells pretreated with 100 µM ryanodine for 45 min are indicated by bold traces; ryanodine was present throughout the experiments. A, Cells were transfected with YC4er. CCh (100 µM) was added at 60 sec, and 3 mM BAPTA was added at 300 sec. Representative traces from 6–10 cells (mean ± SEM) are shown. B, MIN6 cells were loaded with Fura2. CCh (100 µM) was added at 30 sec, and 3 mM BAPTA was added at approximately 150 sec. [Ca2+]c transients from MIN6 cells (mean ± SEM, 15–25 cells per experiment) are shown. Baseline 340/380-nm ratios averaged between 0.35 ± 0.01 to 0.37 ± 0.01 in all experiments. C, Cells loaded with Fura2 were exposed to either 100 µM CCh (left) or 25 mM KCl (right).

 
CICR in INS-1 cells
We also investigated CICR in a different insulin-secreting pancreatic ß-cell line, INS-1 (43). Caffeine caused a marked drop in [Ca2+]er in INS-1 cells expressing YC4er (Fig. 14AGo), as did depolarization with 25 mM KCl (Fig. 14BGo). Fura2-loaded INS-1 cells showed a transient increase in [Ca2+]c on treatment with 20 mM caffeine, and this caffeine effect was blocked by pretreatment with 100 µM ryanodine (Fig. 14CGo). This demonstration of CICR in the INS-1 model supports the notion that CICR is a general mechanism of ER Ca2+ regulation in pancreatic ß-cells.



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  		FIG. 14. Changes in ER and cytoplasmic Ca2+ in INS-1 cells. INS-1 cells expressing YC4er were incubated in KBRS and treated with: A, Caffeine (20 mM); B, 25 mM KCl followed by 1 µM thapsigargin, 3 mM BAPTA, and 500 nM ionomycin (TBI) as indicated. Traces represent mean ± SEM of measurements from 10 cells. C, INS-1 cells were loaded with fura2 and pretreated for 45 min with 100 µM ryanodine (bold trace) or vehicle (fine trace). Caffeine (20 mM) was added as shown, followed by TBI. Starting 340/380-nm ratios were 0.36 ± 0.01 and 0.34 ± 0.01 for untreated and ryanodine-treated cells, respectively. Each trace represents the mean ± SEM of measurements from 15 individual cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have provided direct evidence that Ca2+ is released from the ER in response to a sharp rise in cytoplasmic Ca2+, suggesting that CICR plays a role in the regulation of Ca2+ signals in the pancreatic ß-cell. This is the first report, to our knowledge, in which CICR has been demonstrated in ß-cells by direct observation of changes in [Ca2+]er. The effects of depolarization on release of Ca2+ from the ER were clearly mediated through increases in [Ca2+]c, and not merely a result of depolarization, because VDCC antagonists precluded the effects. As the height of the [Ca2+]c peaks rose with increasing doses of KCl, so did the degree of the drop in [Ca2+]er. This dose-response relationship provides further evidence for depolarization-induced CICR and suggests that the degree of CICR may be regulated. Because the ER cameleon YC4er, with a Kd for Ca2+ of 700 µM, is a rather insensitive reporter, the changes we observed must have represented large changes in [Ca2+]er. The inhibition of CICR by ryanodine was not attributable to an inhibitory effect of the drug on the VDCC, because ryanodine did not alter depolarization-induced 45Ca2+ influx and because ryanodine had no effect on [Ca2+]c when ER Ca2+ stores were depleted with thapsigargin. The presence of CICR in MIN6 cells is further supported by the finding that two RyR agonists, caffeine and 4-chloro-m-cresol, both discharged ER Ca2+.

Lemmens et al. (25) showed that, when ER Ca2+ pools of primary ß-cell cultures from ob/ob mice were depleted by thapsigargin treatment, the plateau phase of the depolarization-induced rise in [Ca2+]c was lowered. This finding led them to speculate that ER Ca2+ can augment cytoplasmic Ca2+ increases caused by influx through VDCCs. We demonstrated a blunting of the [Ca2+]c response to KCl in cells treated with ryanodine, which blocks CICR. This ryanodine effect was erased when cells were treated with thapsigargin to deplete ER Ca2+ stores. Our results, therefore, support the conclusion that CICR contributes to the depolarization response.

ER-targeted cameleon Ca2+ indicators were used in a previous study of Ca2+ dynamics in MIN6 cells (8). In that study, the RyR agonists caffeine and 4-chloro-3-ethylphenol caused a release of Ca2+ from the ER, suggesting that CICR mechanisms could function. These data are concordant with our observations showing that caffeine and 4-chloro-m-cresol release ER Ca2+. In the work of Varadi and Rutter (8), however, high KCl and glucose, which both activate VDCCs, caused [Ca2+]er to increase. Rises in [Ca2+]er in response to KCl and glucose have also been seen in studies using ER-targeted aequorin (21) or furaptra (29) to report changes in [Ca2+]er. These increases in [Ca2+]er may have occurred because the amount of Ca2+ pumped into the ER by SERCA pumps was higher when the concentrations of cytoplasmic Ca2+ and ATP rose in response to depolarization and metabolism of glucose. Our finding that depolarization causes a decrease in [Ca2+]er contradicts these prior observations. Changes in [Ca2+]er will depend on the balance between the rate at which Ca2+ is lost from the ER via CICR and the rate at which cytoplasmic Ca2+ is pumped into the ER by SERCA pumps. We believe that loss of Ca2+ via CICR predominated in our studies because ER Ca2+ stores were maintained at a high level initially. In fact, we have never detected an increase in [Ca2+]er other than a return toward resting levels after the initial decline in [Ca2+]er induced by secretagogues, even in experiments where we added glucose to ryanodine-treated cells. Our procedures did not involve permeabilization of the plasma membrane or Ca2+ starvation, as did earlier studies, and it is possible that such manipulations affected ER Ca2+ stores or CICR mechanisms. Although functional CICR channels have been inferred from pharmacological approaches, actual release of ER Ca2+ in response to increased [Ca2+]c has not been demonstrated previously.

The cameleon studies failed to uncover CICR in response to glucose stimulation in MIN6 cells, even though glucose caused an increase in [Ca2+]c. The failure to see a fall in [Ca2+]er in response to glucose may have been attributable to the small size and slow onset of the [Ca2+]c increase produced by glucose coupled with the low sensitivity of the ER Ca2+reporter. Alternatively, because glucose increases intracellular ATP, the increase in SERCA activity may have simultaneously increased Ca2+ uptake into the ER and obscured any release of Ca2+ through the RyR. It seems unlikely that glucose addition caused a strong activation of SERCA activity in our experiments, however, because we never observed an initial decrease in [Ca2+]c in response to glucose addition. In some model systems, glucose causes a small transient decline in [Ca2+]c that is blocked by thapsigargin and has been attributed to an ATP-dependent increase in SERCA activity (44, 45).

Ryanodine did not alter the drop in [Ca2+]er induced by CCh, although CCh caused the expected increase in [Ca2+]c. There are several possible explanations for the lack of CICR with CCh treatment. The increase in [Ca2+]c caused by CCh was much lower than that stimulated by KCl depolarization and may have been insufficient to activate CICR. Alternatively, IP3 may cause [Ca2+]er to drop to such low levels that the amount of Ca2+ released on activation of RyRs is too small to detect. It is possible that the ER does not release Ca2+ through a ryanodine-sensitive mechanism when ER Ca2+ stores are already being released through the IP3 receptor or that there is direct cross-talk between the IP3 receptor and the RyR or between the RyR and the VDCC.

In conclusion, we have provided direct evidence that influx of Ca2+ through VDCCs is accompanied by ER Ca2+ release. Two cell lines, MIN6 and INS-1, responded to depolarization with ER store depletion, supporting a role for CICR in pancreatic ß-cells. Insulinoma cell lines are not perfect models for normal islets in vivo. Among the inherent limitations of established cell lines are: a modest response to glucose, and a high basal secretory rate. Nonetheless, clonal ß-cell lines have proved to be valuable experimental models; and our findings, in conjunction with other data, provide compelling evidence that CICR occurs in these model systems. At the present time, the few methods available for measuring ER Ca2+ are difficult to adapt to primary cells; but, as these methological problems are overcome, it will be of interest to extend the current studies to normal ß-cells in the islet, where a role for CICR in ß-cell function seems likely.


   Footnotes
 
This work was supported by NIH Grants DK-19974 and -55280 (to P.M.H.) and DK-02439 (to T.K.G.).

Current address for T.K.G.: Department of Veterinary Clinical Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois 61802

BAPTA, 1,2-Bis(2-aminophenoxy)ethane-N, N, N', N'-tetraacetic acid; [Ca2+]c, concentration of cytoplasmic Ca2+; [Ca2+]er, concentration of endoplasmic reticulum Ca2+; CCh, carbamyl choline; CICR, Ca2+-induced Ca2+ release; CPA, cyclopiazonic acid; ER, endoplasmic reticulum; GFP, green fluorescent protein; GLP-1, glucagon-like peptide 1; IP3, 1,4,5-inositol trisphosphate; KBRS, Krebs buffered Ringers solution; Kd, dissociation constant; RyR, ryanodine receptor; SERCA, sarcoplasmic/endoplasmic reticulum Ca2+ ATPase; TBI, thapsigargin, BAPTA, and ionomycin; VDCC, voltage-dependent Ca2+ channel.

Received December 3, 2002.

Accepted for publication April 17, 2003.


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 Introduction
 Materials and Methods
 Results
 Discussion
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