Ca2+-induced Ca2+ Release from the Endoplasmic Reticulum Amplifies the Ca2+ Signal Mediated by Activation of Voltage-gated L-type Ca2+ Channels in Pancreatic beta -Cells*

Raf Lemmens, Olof Larsson, Per-Olof Berggren, and Md. Shahidul IslamDagger

From the Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Endocrine and Diabetes Unit, Karolinska Institutet, Karolinska Hospital, S-171 76 Stockholm, Sweden

Received for publication, October 17, 2000, and in revised form, December 18, 2000


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Stimulus-secretion coupling in pancreatic beta -cells involves membrane depolarization and Ca2+ entry through voltage-gated L-type Ca2+ channels, which is one determinant of increases in the cytoplasmic free Ca2+ concentration ([Ca2+]i). We investigated how the endoplasmic reticulum (ER)-associated Ca2+ apparatus further modifies this Ca2+ signal. When fura-2-loaded mouse beta -cells were depolarized by KCl in the presence of 3 mM glucose, [Ca2+]i increased to a peak in two phases. The second phase of the [Ca2+]i increase was abolished when ER Ca2+ stores were depleted by thapsigargin. The steady-state [Ca2+]i measured at 300 s of depolarization was higher in control cells compared with cells in which the ER Ca2+ pools were depleted. The amount of Ca2+ presented to the cytoplasm during depolarization as estimated from the integral of the increment in [Ca2+]i over time (int Delta [Ca2+]i·dt) was ~30% higher compared with that in the Ca2+ pool-depleted cells. neo-thapsigargin, an inactive analog, did not affect [Ca2+]i response. Using Sr2+ in the extracellular medium and exploiting the differences in the fluorescence properties of Ca2+- and Sr2+-bound fluo-3, we found that the incoming Sr2+ triggered Ca2+ release from the ER. Depolarization-induced [Ca2+]i response was not altered by U73122, an inhibitor of phosphatidylinositol-specific phospholipase C, suggesting that stimulation of the enzyme by Ca2+ is not essential for amplification of Ca2+ signaling. [Ca2+]i response was enhanced when cells were depolarized in the presence of 3 mM glucose, forskolin, and caffeine, suggesting involvement of ryanodine receptors in the amplification process. Pretreatment with ryanodine (100 µM) diminished the second phase of the depolarization-induced increase in [Ca2+]i. We conclude that Ca2+ entry through L-type voltage-gated Ca2+ channels triggers Ca2+ release from the ER and that such a process amplifies depolarization-induced Ca2+ signaling in beta -cells.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Stimulation of pancreatic beta -cells with glucose leads to membrane depolarization and a consequent increase in the activity of the voltage-gated L-type Ca2+ channels in the plasma membrane. The resulting entry of Ca2+ through these channels is an important determinant of the cytoplasmic free Ca2+ concentration ([Ca2+]i)1 necessary for insulin secretion. However, beta -cells, like other cells, also have highly structured Ca2+ stores like the endoplasmic reticulum (ER). These stores are equipped with Ca2+ pumps and Ca2+ release channels and are thus potentially able to modulate depolarization-induced Ca2+ signaling in several ways (1). They may sequester some of the Ca2+ entering through the voltage-gated Ca2+ channels, or they may release additional Ca2+ into the cytoplasm. The latter may be achieved by different mechanisms, e.g. through Ca2+-mediated activation of phosphatidylinositol-specific phospholipase C (PI-PLC) and formation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) or direct gating of the intracellular Ca2+ channels by the incoming Ca2+. The latter phenomenon, called Ca2+-induced Ca2+ release (CICR), is best known in heart muscle, where a small Ca2+ entry through the L-type voltage-gated Ca2+ channels induces a much larger Ca2+ release through ryanodine receptor Ca2+ channels. CICR, however, is not an exclusive property of ryanodine receptors. The other family of intracellular Ca2+ channels, i.e. the inositol 1,4,5-trisphosphate receptors (IP3Rs), as well as the permeability transition pore of mitochondria are also capable of mediating different forms of CICR (2, 3).

beta -Cells express both IP3Rs and ryanodine receptors, the relative proportion of which appears to vary under different conditions (4-6). These Ca2+ channels are located on the ER membrane that constitutes one anatomical Ca2+ pool with luminal continuity (7). The type 3 isoform of the IP3R is the predominant IP3R in ob/ob mouse beta -cells, whereas a ryanodine receptor resembling the type 2 ryanodine receptor of heart is the main ryanodine receptor in these cells (6, 8, 9). Both of these channels can be considered to be Ca2+-gated Ca2+ channels, raising the possibility that depolarization-induced Ca2+ signaling may be amplified by Ca2+ release through these channels. CICR has been considered in beta -cells from indirect evidence (10); but the phenomenon has not been demonstrated in situ, and its role in amplification of Ca2+ signaling in these cells has not been explored. Here we demonstrate that beta -cells possess a mechanism for amplification of depolarization-induced Ca2+ signaling by Ca2+ release from the ER. The ER Ca2+ pools modulate depolarization-induced changes in [Ca2+]i by sequestering Ca2+ from the cytoplasm as well as by releasing Ca2+ into the cytoplasm, a process that is likely to involve CICR.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemicals-- Fura-2/AM, fluo-3 free acid, and fluo-3/AM were from Molecular Probes Europe (Leiden, The Netherlands). Thapsigargin was from Life Technologies, Inc. and Calbiochem. neo-thapsigargin was a generous gift from Drs. S. B. Christensen and M. Treiman (Royal Danish School of Pharmacy, Copenhagen, Denmark). Ryanodine (98% pure) and ET-18-OCH3 (1-octadecyl-2-methyl-rac-glycero-3-phosphocholine) were from Calbiochem. U73122 (1-[6-([(17beta )-3-methoxyestra-1,3,5(10)-trien-17-yl]amino)hexyl]-1H-pyrrole-2,5-dione) was a gift from Dr. J. E. Bleasdale (The Upjohn Co., Kalamazoo, MI). 2-Aminoethoxydiphenyl borate (also called diphenylboric acid 2-aminoethyl ester) was a gift from Dr. T. Maruyama (Ono Pharmaceutical Co., Minase Research Institute, Osaka, Japan) and was also bought from Sigma. Neomycin sulfate was from the Upjohn Co. Cyclopiazonic acid and 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate were from Sigma. Xestospongin C was a gift from Dr. Kobayashi, Osaka University, and was also bought from Calbiochem.

Isolation of Pancreatic Islets and Preparation of beta -Cells-- Islets from fasting adult ob/ob mice from a local colony were isolated by collagenase digestion and dispersed by shaking in Ca2+- and Mg2+-deficient medium (7). Cells were cultured on glass coverslips for 1-2 days in RPMI 1640 medium supplemented with fetal calf serum (10%, v/v), penicillin (100 IU/ml), and streptomycin (100 µg/ml).

Measurement of [Ca2+]i by Microfluorometry-- Cells were incubated in RPMI 1640 medium supplemented with 0.1% bovine serum albumin and 0.6 µM fura-2/AM for 35 min at 37 °C. Cells were then incubated for an additional 10 min in basal medium containing 125 mm NaCl, 5.9 mM KCl, 1.2 mM MgCl2, 1.28 mM CaCl2, 25 mM HEPES, 3 mM glucose, and 0.1% bovine serum albumin (pH 7.4). Coverslips were mounted as the bottom of a perifusion chamber on the stage of an inverted epifluorescence microscope (Zeiss Axiovert 35M). The superfusion chamber was designed to allow rapid exchange of fluids, and complete exchange of fluids took place in <3 s. The stage was thermostatically controlled to maintain a temperature of 37 °C in the perifusate inside the chamber. The microscope was connected to a SPEX Fluorolog-2 CM1T11I system for dual wavelength excitation fluorometry. The excitation wavelengths generated by two monochromators were directed to the cell by a dichroic mirror. The emitted light selected by a 510-nm filter was monitored by a photomultiplier. The excitation wavelengths were alternated at a frequency of 1 Hz, and the duration of data collection at each wavelength was 0.33 s. The emission at the excitation wavelength of 340 nm (F340) and that of 380 nm (F380) were used to calculate the fluorescence ratio (R340/380). Single cells or small clusters of cells, isolated optically by means of a diaphragm, were studied by using a 40× 1.3 NA oil immersion objective (Zeiss, Plan Neofluar). Background fluorescence was measured after quenching fura-2 fluorescence with manganese and was subtracted from the traces before calculation of [Ca2+]i. [Ca2+]i was calculated from R340/380 according to Grynkiewicz et al. (11). Maximum and minimum fluorescence ratios were determined in separate experiments using thin films of external standards containing 10 µM fura-2 and 2 M sucrose in intracellular-like buffer containing 10 µM fura-2 free acid and either 2 mM Ca2+ or no Ca2+ in the presence of 2 mM EGTA. The Kd for the Ca2+·fura-2 complex was taken as 224 nM. To compensate for variations in output light intensity from the two monochromators, all experiments included a fluorescence ratio where both monochromators were set at 360 nm.

Fluo-3 Fluorescence Measurements-- For comparing the fluorescence properties of Ca2+- or Sr2+-bound fluo-3, we dissolved 1 µM fluo-3 free acid in buffer containing 145 mM KCl and 20 mM HEPES (pH 7.2). The buffer was then divided in to two portions: to one we added SrCl2 (1 mM), and to the other CaCl2 (1 mM). Emission spectra were measured at 490 nm excitation using a SPEX Fluorolog-2 CM1T11I system. Scanning was performed in a quartz cuvette containing a 1-ml solution at 20 °C. Cells were loaded with fluo-3 by incubation with 0.5 µM fluo-3/AM for 35 min, followed by incubation in basal medium for another 10 min. Fluorescence was measured at 490 nm excitation, and emitted light filtered by a 515-565-nm band-pass filter was recorded by a photomultiplier.

Electrophysiological Recordings-- Voltage-gated Ca2+ currents were recorded using the whole-cell mode of the patch-clamp technique (12). Pipettes were prepared from borosilicate glass capillary tubes, coated with Sylgard resin (Dow Corning) near the tips, and fire-polished and had resistances of 2-6 mega-ohms. Cells were washed with a solution composed of 138 mM NaCl, 5.6 mM KCl, 1.2 mM MgCl2, 10 mM CaCl2, 10 mM tetraethylammonium chloride, and 5 mM HEPES (pH 7.4). The pipette solution contained 150 mM N-methyl-D-glucamine, 110 mM HCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM EGTA, 3 mM MgATP, and 5 mM HEPES (pH 7.15). N-Methyl-D-glucamine was substituted for K+ in the pipette solution to prevent outwardly directed K+ currents. Currents were recorded using an Axopatch 200 amplifier (Axon Instruments, Inc., Foster City, CA). Voltage steps were generated, digitized, and stored using the program pClamp (Axon Instruments, Inc.) and Labmaster ADC (Scientific Solutions). The current responses were filtered at 2 kHz. The pulse protocol is given in the figure legends.

Statistical Analysis-- Numerical data are presented as means ± S.E. Statistical significance was judged by Student's t test for unpaired data.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

beta -Cells perifused with basal medium containing 3 mM glucose responded with an increase in the average [Ca2+]i upon depolarization with 30 mM KCl (Fig. 1). Consistent with our previous reports (13), the [Ca2+]i increase was dependent on the presence of extracellular Ca2+ and was abolished by nimodipine, a specific blocker of the L-type voltage-gated Ca2+ channels. These results suggest that in cultured mouse beta -cells and under our experimental conditions, depolarization-induced Ca2+ entry occurs entirely through the L-type voltage-gated Ca2+ channels and that depolarization per se does not induce detectable Ca2+ release from internal stores. Depolarization by KCl yielded a rise in [Ca2+]i, the pattern of which varied from cell to cell. In all cells, however, [Ca2+]i increased in two phases, an initial rapid phase followed by a slower increase to the peak (Fig. 1A). On examination of a large number of cells, three patterns of [Ca2+]i increase could be discerned (Fig. 1). The most common was one where a rapid increase in [Ca2+]i changed into a slower phase (Fig. 1A). In 6 out of 19 cells, the two phases were separated by a transient dip in [Ca2+]i after the initial rapid phase (Fig. 1B). In two other cells, the second phase of the increase in [Ca2+]i consisted of a single Ca2+ transient on elevated [Ca2+]i. In some experiments, we used beta -cells obtained from Wistar rats instead of ob/ob mice, and the results were similar.


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Fig. 1.   Patterns of [Ca2+]i changes in pancreatic beta -cells during sustained depolarization. Fura-2-loaded cultured mouse beta -cells were perifused with basal medium containing 3 mM glucose and depolarized by rapid application of 30 mM KCl during the period indicated by the horizontal bars. Following depolarization, [Ca2+]i increased to a peak in two phases. The pattern of [Ca2+]i increase shown in A occurred in 11 out of 19 cells. In some cells (B), the two phases were separated by a transient dip after the first rapid phase of rise in [Ca2+]i (6 out of 19 cells). In two cells, the depolarization-induced increase in [Ca2+]i was accompanied by a Ca2+ transient on an elevated [Ca2+]i (C).

A biphasic increase in [Ca2+]i suggested the possibility that multiple sources of Ca2+ may be involved in depolarization-induced Ca2+ signaling. To test whether the ER Ca2+ stores contributed to the depolarization-induced changes in [Ca2+]i, we examined [Ca2+]i responses in cells where the ER Ca2+ stores were depleted by thapsigargin, a specific inhibitor of SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPases) (14). Incubation of cells with 250 nM thapsigargin for 35 min depleted the ER Ca2+ pools as indicated by lack of response to carbachol. The resting [Ca2+]i and membrane potential in thapsigargin-treated cells were same as in the controls (Figs. 2C and 3A), indicating that capacitative Ca2+ entry was unlikely to confound results under these experimental conditions. In ER Ca2+ pool-depleted cells, the [Ca2+]i response upon depolarization differed from that in control cells with regard to several aspects. In thapsigargin-treated cells, KCl increased [Ca2+]i with a faster time course, and the decay phase around the peak was also faster (Fig. 2C). The time courses of increases in [Ca2+]i from the basal level to the peak subsequent to depolarization in control and thapsigargin-treated cells are illustrated in Fig. 2A. In this figure, where data are presented as semilogarithmic plots, the time course of the increase in [Ca2+]i in control cells clearly deviates from a single exponential. Most strikingly, thapsigargin treatment abolished the slower second phase of the increase in [Ca2+]i seen in the control cells (Fig. 2, A and C). [Ca2+]i increased more rapidly in thapsigargin-treated cells than in control cells. The maximum rates of increase in [Ca2+]i, calculated as the differences between neighboring points, were 90 ± 14 and 45 ± 6 nM/s in the thapsigargin-treated and control cells, respectively (p = 0.006, n = 28). The times required for [Ca2+]i to rise from basal levels to the peaks were 19 ± 4 and 85 ± 12 s in thapsigargin-treated and control cells, respectively (p < 0.001, n = 30) (Fig. 2B). Depolarization-induced peak [Ca2+]i was slightly higher in the thapsigargin-treated cells, although the difference was not statistically significant (485 ± 50 and 403 ± 30 nM in thapsigargin-treated and control cells, respectively; n = 26). For comparison of [Ca2+]i response in the two groups, we calculated the integral of the increment in [Ca2+]i over time (int Delta [Ca2+]i·dt) as an estimate of the amount of Ca2+ presented to the cytoplasm during depolarization. A period of 300 s was chosen for calculating the Ca2+ integral because during sustained depolarization by KCl, [Ca2+]i returned to an elevated steady-state level in most experiments by this time. The steady-state [Ca2+]i measured at 300 s of depolarization was significantly lower in thapsigargin-treated cells (171 ± 16 and 233 ± 11 nM in thapsigargin-treated and control cells, respectively; p = 0.003, n = 26). The time integral of the [Ca2+]i increment during 300 s of stimulation by KCl was ~30% higher in the control cells compared with that in the Ca2+ pool-depleted cells (p = 0.002, n = 26) (Fig. 2D). In some experiments, we treated cells with a lower concentration of thapsigargin (e.g. 50 nM). This concentration of thapsigargin depleted ER Ca2+ pools almost completely, but such depletion was achieved less consistently. However, a qualitatively similar, although smaller effect on [Ca2+]i response was seen also when cells were treated with 50 nM thapsigargin. In some other experiments, we treated cells with 10 µM cyclopiazonic acid, a reversible inhibitor of SERCA, for 30 min and depolarized the cells with KCl in the continued presence of the substance. The effects of treatment with cyclopiazonic acid were similar to those obtained with thapsigargin.


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Fig. 2.   Effect of depletion of ER Ca2+ pools on depolarization-induced [Ca2+]i response. ER Ca2+ stores were depleted by incubating the cells for 35 min with 250 nM thapsigargin. Cells were depolarized by 30 mM KCl. A, the time course of rise in [Ca2+]i from the basal to the peak level upon depolarization is plotted on a semilogarithmic scale. Three traces each for controls () and thapsigargin (Tg)-treated cells (open circle ) are illustrated. The traces are representative of 15 experiments with control cells and 15 experiments with thapsigargin-treated cells. B, shown is the time taken for the increase in [Ca2+]i from the basal level to the peak in control and thapsigargin-treated cells. C, changes in [Ca2+]i during 300 s of depolarization by 30 mM KCl in control and thapsigargin-treated cells are illustrated. Traces are representative of 13 experiments done with thapsigargin-treated cells and 13 with control cells. D, the time integral of the increase in [Ca2+]i above the base line during 300 s of depolarization by KCl was significantly higher in control cells compared with thapsigargin-treated cells . Data are derived from 13 experiments in control cells and 13 thapsigargin-treated cells as illustrated in C.

We tested whether the reduction in depolarization-induced [Ca2+]i response in thapsigargin-treated cells could be due to inhibition of L-type voltage-gated Ca2+ channels by the compound, as has been reported in other cells (15). As shown in Fig. 3A, depolarization-induced whole-cell Ca2+ currents measured by the patch-clamp technique in cells treated with a low concentration of thapsigargin (i.e. 250 nM) were not significantly different from those recorded in controls. To test further if some other nonspecific effects of thapsigargin could account for our results, we treated cells with an analog of the compound epimerized at C-8 (neo-thapsigargin) (16). When used at low concentrations, this analog of thapsigargin is virtually inactive as an inhibitor of SERCA (17). As shown in Fig. 3B, neo-thapsigargin (250 nM for 35 min) did not deplete the ER Ca2+ pools and also did not alter the pattern of the depolarization-induced increase in [Ca2+]i (cf. Fig. 1 and Fig. 2C). It was a possibility that thapsigargin treatment and the consequent increase in [Ca2+]i caused enhancement of plasma membrane Ca2+-ATPase activity, thus accounting for the less pronounced [Ca2+]i response seen in these cells (18). To test this, we increased [Ca2+]i by activating the reverse mode of the Na+/Ca2+ exchanger by substituting Na+ with N-methyl-D-glucamine in the perifusion medium. This maneuver caused a modest increase in [Ca2+]i, the magnitude of which was not significantly different in thapsigargin-treated cells compared with the untreated controls (data not shown).


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Fig. 3.   Altered [Ca2+]i response in thapsigargin-treated cells is not due to nonspecific effects of the substance. A, treatment of cells with a low concentration of thapsigargin (250 nM) for 35 min did not alter the magnitude of voltage-dependent Ca2+ currents through Ca2+ channels in the plasma membrane. Cells were depolarized in 10-mV steps (100 ms) to potentials between -60 and +50 mV from a holding potential of -70 mV. The upper traces show examples of currents in the presence and absence of thapsigargin. The lower graph shows compiled data on the I-V relationship. B, neo-thapsigargin, an inactive isomer of thapsigargin, did not alter depolarization-induced [Ca2+]i response. Cells were incubated for 35 min in 250 nM neo-thapsigargin. Depolarization by KCl resulted in a biphasic increase in [Ca2+ ]i as in control cells. ER Ca2+ pools were not depleted by neo-thapsigargin since carbachol (CCh) released Ca2+ from the ER. Traces are representative of experiments repeated at least three times. pF, picofarads.

The results described above suggest the possibility that Ca2+ entering through the L-type voltage-gated Ca2+ channels might act as a trigger, giving rise to further Ca2+ release from the ER. We intended to visualize the trigger Ca2+ and the released Ca2+ more directly. This could be done by using Sr2+ as a Ca2+ surrogate and by exploiting the differences in the fluorescence properties of Ca2+- and Sr2+-bound fluo-3. We compared the fluorescence spectral properties of fluo-3 in the presence of Ca2+ and Sr2+. Fig. 4A shows the emission spectra of fluo-3 between 500- and 560-nm wavelengths in the presence of saturating concentrations of Ca2+ or Sr2+. As shown, fluo-3 yielded much less fluorescence when it bound Sr2+ compared with when it bound Ca2+. In the experiments illustrated in Fig. 4 (B-D), we used fluo-3/AM instead of fura-2/AM to load the cells. In these experiments, we omitted Ca2+ from the perifusion medium and instead added 1 mM Sr2+ to the medium. Under these conditions, when the cells were depolarized by KCl, there was initially a modest increase in fluorescence presumably due to Sr2+ entry and the resulting increase in [Sr2+]i. Superimposed on this fluorescence, there was a large but transient increase in fluorescence, which was probably caused by Ca2+ release from intracellular stores (Fig. 4B). To ascertain whether the Ca2+ transients triggered by Sr2+ were due to Ca2+ release from the ER, we depleted the ER Ca2+ stores with thapsigargin and repeated similar experiments. As shown in Fig. 4C, in thapsigargin-treated cells, despite depolarization-induced Sr2+ entry, there was no Ca2+ spike as seen in the untreated cells. Sr2+ was unable to trigger Ca2+ release also when the common ER Ca2+ pools were depleted by carbachol prior to depolarization by KCl (Fig. 4D).


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Fig. 4.   Differences in fluorescence properties of fluo-3 complexes with Sr2+ and Ca2+ used to visualize Ca2+ release from ER. A, shown are the fluorescence emission spectra of fluo-3 in the presence of Ca2+ or Sr2+. Spectra were obtained in HEPES/KCl buffer containing 1 µM fluo-3 free acid and 1 mM Ca2+ or Sr2+. The traces are representative of experiments repeated twice. B, cells were loaded with fluo-3 and perifused with extracellular medium containing 1 mM Sr2+ instead of Ca2+. Depolarization of cells by 30 mM KCl in the presence of extracellular Sr2+ resulted in an initial small increase in fluorescence followed by large transient increases in fluorescence. The latter is caused by Ca2+ released from the ER since it was abolished when the ER Ca2+ pools were first depleted by thapsigargin (Tg; C) or prior application of carbachol (10 µM) (D). The traces are representative of experiments repeated at least three times.

These results suggest that during sustained depolarization and in association with Ca2+ entry through the L-type voltage-gated Ca2+ channels, there is a release of Ca2+ from the ER. Such a Ca2+ release could be mediated by several mechanisms. The first would involve activation of PI-PLC by Ca2+ entering through the voltage-gated Ca2+ channels, leading to formation of Ins(1,4,5)P3 and the consequent activation of IP3Rs. The second would involve direct gating of IP3Rs by Ca2+ in the presence of a basal or slightly elevated level of the trisphosphate. The third would involve activation of the ryanodine receptors by Ca2+. To determine whether activation of PI-PLC by Ca2+ was involved, we tried several inhibitors of the enzyme, e.g. neomycin, the ether lipid ET-18-OCH3 (19), and 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate (20). However, we found that these inhibitors themselves alter [Ca2+]i homeostasis in beta -cells by nonspecific mechanisms unrelated to the inhibition of PI-PLC. Neomycin inhibited L-type voltage-gated Ca2+ channels as studied by the whole-cell mode of the patch-clamp technique, an observation consistent with a previous report (21). When applied to the resting beta -cells, ET-18-OCH3 and 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate increased [Ca2+]i by unknown mechanisms. These substances were therefore not suitable as PI-PLC inhibitors in intact beta -cells. We then tested another commonly used inhibitor of PI-PLC, U73122. This substance, despite reported nonspecific effects, appeared suitable for inhibiting PI-PLC in beta -cells. We perifused beta -cells with 10 µM U73122 for 300 s and then depolarized the cell by KCl in the continued presence of the inhibitor. U73122 itself did not affect [Ca2+]i in beta -cells. As shown in Fig. 5A, U73122 did not inhibit the biphasic increase in [Ca2+]i during KCl depolarization (cf. Fig. 5B). Under these conditions, U73122 inhibited PI-PLC as evidenced by lack of [Ca2+]i response to carbachol in separate experiments. These results indicate that amplification of depolarization-induced Ca2+ signaling does not require concomitant activation of PI-PLC by the incoming Ca2+. To directly test if activation of IP3Rs by Ins(1,4,5)P3 or Ca2+ was involved, we intended to use xestospongin C and 2-aminoethoxydiphenyl borate, membrane-permeable agents that have been reported to inhibit IP3Rs in neuronal cells (22). However, in control experiments, we found that xestospongin C (20 µM) or 2-aminoethoxydiphenyl borate (up to 300 µM) (23) did not inhibit Ca2+ release by Ins(1,4,5)P3-forming agonists like carbachol or ADPbeta s in beta -cells. These agents thus did not appear suitable as membrane-permeable inhibitors of IP3Rs in beta -cells.


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Fig. 5.   Effect of a PI-PLC inhibitor (U73122) on the depolarization-induced increase in [Ca2+]i in beta -cells. Fura-2-loaded single beta -cells were first perifused by 10 µM U73122 (A) or 0.1% Me2SO (DMSO; solvent for U73122) (B) for 300 s. Cells were then depolarized by applying KCl (30 mM) until [Ca2+]i increased to its peak. In both cases, [Ca2+]i increased to a peak in two phases. Data are representative of experiments repeated at least three times with similar results.

To test if ryanodine receptors were involved in amplifying depolarization-induced Ca2+ signaling, we used ryanodine, ruthenium red, and caffeine. We incubated cells with 100 µM ryanodine for 45 min. Such treatment is expected to lock the ryanodine receptors in a subconductance state and thus deplete the ryanodine-sensitive ER Ca2+ pools to a variable extent. The effect of ryanodine treatment on depolarization-induced Ca2+ response was small. When ryanodine-treated cells were depolarized by KCl, the second phase of the [Ca2+]i increase was diminished (Fig. 6B) as compared with that in untreated controls (Fig. 6A). Consistent with this, the time taken for [Ca2+]i to rise from the basal level to the peak was significantly reduced in ryanodine-treated cells compared with the untreated controls (52 ± 12 and 146 ± 27 s, respectively; p < 0.05, n = 7) (Fig. 6C). Low concentration of ruthenium red (10 µM), an inhibitor of ryanodine receptors, reduced depolarization-induced [Ca2+]i response (Fig. 6D). We then examined whether agents that sensitize ryanodine receptors can increase depolarization-induced Ca2+ response. Caffeine sensitizes ryanodine receptors to Ca2+, and from previous studies, we know that in situ activation of ryanodine receptors by caffeine requires cAMP-dependent phosphorylation (5). When cells were depolarized in the presence of 3 mM glucose and forskolin (5 µM) plus caffeine (5 mM), a dramatic increase in [Ca2+]i response in the form of Ca2+ spikes was observed (Fig. 7A). Such an increased Ca2+ response could be due to increased Ca2+ entry through L-type voltage-gated Ca2+ channels caused by cAMP-dependent phosphorylation of the channel. But our control experiments showed that this was not the case since forskolin alone did not have a similar effect under these experimental conditions (Fig. 7B).


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Fig. 6.   Effect of ryanodine and ruthenium red on depolarization-induced increase in [Ca2+]i in beta -cells. A, fura-2-loaded single beta -cells were depolarized by 30 mM KCl during the period indicated by the horizontal line. The trace shows a control experiment with a typical biphasic increase in [Ca2+]i to its peak. B, cells were incubated for 45 min with 100 µM ryanodine and depolarized by KCl. The second phase of the [Ca2+]i increase was diminished by ryanodine treatment. C, shown is the time taken for the increase in [Ca2+]i from the basal level to the peak in control and ryanodine-treated cells (p < 0.05, n = 7). D, [Ca2+]i response was lower when cells were depolarized by KCl in the presence of 10 µM ruthenium red (RR), a blocker of the ryanodine receptor.


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Fig. 7.   Sensitization of the ryanodine receptor enhances the depolarization-induced [Ca2+]i increase in beta -cells. Fura-2-loaded beta -cells were perifused with basal buffer containing 3 mM glucose. A, cells were depolarized by KCl in the presence of 5 µM forskolin and 5 mM caffeine. B, shown are the results of a control experiment in which cells were depolarized by KCl in the presence of 5 µM forskolin only. Note that the scales are different in A and B. Traces are representative of at least three experiments with similar results.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasma membrane depolarization and increase in [Ca2+]i are two key events in stimulus-secretion coupling of pancreatic beta -cells. It is generally thought that depolarization-induced [Ca2+]i increase in beta -cells is solely due to Ca2+ entry through the voltage-gated Ca2+ channels in the plasma membrane. In many electrically excitable cells, however, Ca2+ entering through the voltage-gated Ca2+ channels activates adjacent intracellular Ca2+ channels (CICR). In such cells, depolarization-induced [Ca2+]i increase is a result of Ca2+ being presented to the cytoplasm through two types of channels, i.e. the plasma membrane Ca2+ channels and the ER Ca2+ channels. beta -Cells possess the requisite molecular components required for mediating CICR (5, 10). However, the issue of whether ER Ca2+ stores and the associated Ca2+ channels participate in the depolarization-induced Ca2+ signaling in intact beta -cells has remained an open question. Thapsigargin, a tool for studying involvement of ER Ca2+ stores in cellular processes, has made it possible to address this question (24). Using this approach, we now demonstrate that the ER Ca2+ stores modulate depolarization-induced Ca2+ signaling in two ways. In our study, the rate of increase in [Ca2+]i following depolarization was significantly faster in thapsigargin-treated cells compared with controls. This suggests that following Ca2+ entry through the voltage-gated Ca2+ channels, the ER Ca2+-ATPase immediately participates in removing Ca2+ from the cytoplasm into the ER. This rapid sequestration of Ca2+ into the ER can retard the rate of increase in [Ca2+]i and limit the peak [Ca2+]i during depolarization. Such effects may have implications in the prevention of short-term and long-term toxic effects of [Ca2+]i on beta -cells. On the other hand, Ca2+ uptake into the ER may have important consequences in loading the latter compartment. This can ensure generation of a larger Ca2+ flux from the ER, thereby prolonging the Ca2+ transient and amplifying [Ca2+]i-dependent signaling (25).

The other important finding of our study is that depolarization-induced Ca2+ signaling in beta -cells is amplified by release of the ion from the intracellular stores. The Ca2+ stores that participate in this process are the ones associated with the ER. This is evident from the fact that during depolarization, the steady-state [Ca2+]i as well as the integral of the increment in [Ca2+]i over time were significantly higher in control cells compared with cells in which the ER Ca2+ pools were depleted by thapsigargin. The time integral of [Ca2+]i response was higher in the control cells compared with the thapsigargin-treated cells, despite the fact that, in the latter, the ER Ca2+ pump was inhibited, and ER could not contribute to the removal of Ca2+ entering into the cytoplasm through the voltage-gated Ca2+ channels. To demonstrate more directly that Ca2+ entering through the voltage-gated Ca2+ channels can activate ER Ca2+ channels in beta -cells, we substituted Sr2+ for Ca2+ in the extracellular medium. The rationale for this approach is that Sr2+ can enter through the voltage-gated Ca2+ channels and can activate ryanodine receptors (26). But whereas Ca2+ yields large fluorescence upon binding to fluo-3, Sr2+ does not (Fig. 4A). Using this approach, we could demonstrate that Sr2+, entering through the voltage-gated Ca2+ channels, induces release of Ca2+ from the ER. Thus, our results show that during depolarization, the net increase in [Ca2+]i in beta -cells is a result of Ca2+ entry through voltage-gated Ca2+ channels and Ca2+ release from the ER, suggesting that the two processes may be coupled. The succession of two events, i.e. Ca2+ uptake into and release from the ER, may thus play a role in modulating the magnitude and spatiotemporal aspects of depolarization-induced [Ca2+]i signaling in beta -cells.

The mechanisms that couple Ca2+ entry through the voltage-gated L-type Ca2+ channels to Ca2+ release from the ER may involve multiple ER Ca2+ channels. One view is that depolarization and the resulting increase in [Ca2+]i activate PI-PLC, leading to generation of Ins(1,4,5)P3, which by itself activates IP3Rs, causing a further increase in [Ca2+]i. This view originates from the facts that a minimal level of Ca2+ is essential for activation of PI-PLC (27, 28) and that Ca2+ increases PI-PLC activity in mouse islet homogenates (28). In this view, an increase in the concentration of Ins(1,4,5)P3 (as opposed to an increase in [Ca2+]) is the predominant mechanism that couples Ca2+ entry to Ca2+ release. However, the relative roles played by any postulated increase in the concentration of Ins(1,4,5)P3 versus an increase in [Ca2+] in this coupling process merit closer examination. In intact cells, KCl depolarization leads to little or only a modest and transient (~30 s) increase in PI-PLC activity (29-32). This is particularly true for mouse islets, which express low levels of Ca2+-sensitive PI-PLC isoforms such as PI-PLCdelta 1 (29, 33). Moreover, studies that have demonstrated increased PI-PLC activity in islets during KCl depolarization were performed using suspensions of islets in static incubations. Under such conditions, depolarization-induced Ca2+-dependent exocytosis leads to accumulation of components of secretory granules, e.g. ATP, in the medium, which may stimulate PI-PLC by activating specific receptors. Stimulation of PI-PLC activity seen in these assays may thus be partly due to autocrine or paracrine stimulation of receptors linked to PI-PLC. Thus, these protocols are not suitable to test whether depolarization-induced increases in [Ca2+]i per se can directly stimulate PI-PLC in intact beta -cells in the absence of ligand binding. Studies in other cells demonstrate that a physiological increase in [Ca2+]i does not directly activate PI-PLC (34), but may potentiate activation of the enzyme when PI-PLC-linked receptors are occupied by appropriate ligands (35). In our experiments, in which we used single mouse beta -cells and a system for rapid washout of medium by continuous perifusion, the likelihood of autocrine or paracrine interactions was minimal. It is therefore likely that, under our experimental conditions, a KCl-induced rise in [Ca2+]i increased cellular Ins(1,4,5)P3 level only minimally. The molecular mechanisms involved in the opening of IP3Rs are complex. In addition to Ins(1,4,5)P3, Ca2+ plays a fundamental role in this process. From studies on Ins(1,4,5)P3-induced Ca2+ release in different cells, it is known that Ca2+ and Ins(1,4,5)P3 are coactivators of the IP3R (36), i.e. the receptor must bind both of these agonists before the associated channel opens (37). From these considerations, it appears likely that Ca2+ entering through the voltage-gated Ca2+ channels can directly trigger IP3R, apparently in the context of minimal elevation of the trisphosphate. In this scenario, the IP3R is viewed to operate mainly as a CICR channel. In our experiments, U73122, an inhibitor of PI-PLC, did not reduce amplification of depolarization-induced Ca2+ signaling. This observation lends support to the notion that activation of PI-PLC by Ca2+ is not essential for the amplification of depolarization-induced Ca2+ signaling.

Among the IP3Rs, ob/ob beta -cells express mainly the type 3 isoform (IP3R-3) (6). This isoform of IP3R is 10 times less sensitive to the trisphosphate compared with IP3R-1 and even less sensitive than IP3R-2 (38). Given that Ins(1,4,5)P3 and Ca2+ are co-agonists of IP3Rs and that IP3R-3 is only poorly sensitive to the trisphosphate alone, it is likely that, under physiological conditions, the role of Ca2+ as a trigger of IP3R-3 is of crucial importance. In other words, IP3R-3 can operate as a CICR channel much like the ryanodine receptors, albeit requiring assistance from a basal or minimally elevated level of the trisphosphate. Of the three ryanodine receptors, beta -cells appear to express only the type 2 isoform, which typically mediates CICR in heart (5, 6, 9). The level of expression of ryanodine receptors in our ob/ob beta -cells is low compared with that in heart (5). Despite this, depolarization-induced [Ca2+]i response was dramatically enhanced by caffeine when the xanthine drug was applied together with a cAMP-elevating agent, forskolin. It may be noted that this enhancement could not be attributed to cAMP-dependent phosphorylation of L-type voltage-gated Ca2+ channels since forskolin alone did not have significant effects on [Ca2+]i response. cAMP-dependent phosphorylation is required for in situ activation of ryanodine receptors in beta -cells (5). Protein kinase A phosphorylation relieves the inhibition of the ryanodine receptor by intracellular Mg2+ and promotes dissociation of FKBP12.6, events that facilitate channel activation (39, 40). These results provide good evidence that type 2 ryanodine receptors participate in amplification of depolarization-induced Ca2+ signaling in beta -cells, but do not exclude the roles of IP3Rs in the process. Since Ca2+ can gate both of these channels, it is likely that both types of channels participate in mediating CICR. The fact that ruthenium red and high concentrations of ryanodine only partially blocked the second phase of depolarization-induced [Ca2+]i response further supports that both ryanodine receptors and IP3Rs may be involved in the process.

We demonstrate that in addition to the voltage-gated Ca2+ channels in the plasma membrane, the ER Ca2+ stores participate in generating depolarization-induced Ca2+ signaling in beta -cells. The latter does not act only as a Ca2+ sink, but rather plays important roles in amplifying the depolarization-induced Ca2+ signaling by CICR. We have demonstrated amplification of Ca2+ signaling in cultured beta -cells by using EGTA-based Ca2+ indicators that act as mobile buffers and that have the potential to interfere with the process of CICR. It is likely that the amplification process is more pronounced in vivo. Such amplified Ca2+ signaling may have physiological relevance in mediating cellular processes that require high [Ca2+]i.

    FOOTNOTES

* This work was supported in part by Swedish Medical Research Council Grants K200-32X-13469-01A, 72X-09890, 72X-00034, 72XS-12708, and 72X-09891; Swedish Natural Science Research Council Grant U-AA/ST 11616-302l; the Novo Nordisk Foundation; the Swedish Diabetes Association; the Swedish Society of Medicine; and the Karolinska Institutet.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Recipient of a career development award from the Juvenile Diabetes Research Foundation. To whom correspondence should be addressed. Tel. and Fax: 46-8-6731832; E-mail: Shahidul.Islam@molmed.ki.se.

Published, JBC Papers in Press, January 3, 2001, DOI 10.1074/jbc.M009463200

    ABBREVIATIONS

The abbreviations used are: [Ca2+]i, cytoplasmic free Ca2+ concentration; ER, endoplasmic reticulum; PI-PLC, phosphatidylinositol-specific phospholipase C; Ins(1, 4,5)P3, inositol 1,4,5-trisphosphate; CICR, Ca2+-induced Ca2+ release; IP3R, inositol 1,4,5-trisphosphate receptor; AM, acetoxymethyl ester; ADPbeta S, adenosine 5'-O-(2-thiodiphosphate).

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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