Contribution of the endoplasmic reticulum to the glucose-induced [Ca2+]c response in mouse pancreatic islets

Abdelilah Arredouani, Jean-Claude Henquin, and Patrick Gilon

Unité d'Endocrinologie et Métabolisme, University of Louvain Faculty of Medicine, UCL 55.30, B-1200 Brussels, Belgium


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Thapsigargin (TG), a blocker of Ca2+ uptake by the endoplasmic reticulum (ER), was used to evaluate the contribution of the organelle to the oscillations of cytosolic Ca2+ concentration ([Ca2+]c) induced by repetitive Ca2+ influx in mouse pancreatic beta -cells. Because TG depolarized the plasma membrane in the presence of glucose alone, extracellular K+ was alternated between 10 and 30 mM in the presence of diazoxide to impose membrane potential (MP) oscillations. In control islets, pulses of K+, mimicking regular MP oscillations elicited by 10 mM glucose, induced [Ca2+]c oscillations whose nadir remained higher than basal [Ca2+]c. Increasing the depolarization phase of the pulses while keeping their frequency constant (to mimic the effects of a further rise of the glucose concentration on MP) caused an upward shift of the nadir of [Ca2+]c oscillations that was reproduced by raising extracellular Ca2+ (to increase Ca2+ influx) without changing the pulse protocol. In TG-pretreated islets, the imposed [Ca2+]c oscillations were of much larger amplitude than in control islets and occurred on basal levels. During intermittent trains of depolarizations, control islets displayed mixed [Ca2+]c oscillations characterized by a summation of fast oscillations on top of slow ones, whereas no progressive summation of the fast oscillations was observed in TG-pretreated islets. In conclusion, the buffering capacity of the ER in pancreatic beta -cells limits the amplitude of [Ca2+]c oscillations and may explain how the nadir between oscillations remains above baseline during regular oscillations or gradually increases during mixed [Ca2+]c oscillations, two types of response observed during glucose stimulation.

cytosolic Ca2+ concentration oscillations; endoplasmic reticulum; pancreatic beta -cell; thapsigargin


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

GLUCOSE STIMULATES INSULIN SECRETION by producing, triggering, and amplifying signals in pancreatic beta -cells (22). The triggering signal, an increase in cytosolic free Ca2+ concentration ([Ca2+]c), is the outcome of the following sequence of events: the acceleration of metabolism augments the ATP/ADP ratio, which closes ATP-sensitive K+ channels (K+-ATP channels) in the plasma membrane, leading to membrane depolarization, opening of voltage-dependent calcium channels, and Ca2+ influx (5, 22, 39).

Although single-cell studies have been extremely informative on the regulation of ionic currents and Ca2+ handling in beta -cells (4, 10, 20), intact islets, in particular mouse islets, are a more physiological preparation, permitting comparison of the changes in membrane potential and [Ca2+]c (13, 38, 43). The validity of the model is attested by the similarity of the changes in electrical activity and [Ca2+]c in vitro and in vivo (15). Stimulation by a sudden increase in the glucose concentration typically induces an initially small lowering of [Ca2+]c, followed by a biphasic rise consisting of a pronounced first peak, lasting 1-3 min, and a second phase consisting of a sustained elevation with superimposed [Ca2+]c oscillations (3, 13, 45). These oscillations are regular and rapid (frequency of ~2.5/min), regular and slow (frequency of ~0.2/min), or mixed, with rapid oscillations superimposed on slow ones (23). They are associated with periodic depolarizations of the beta -cell membrane (10, 13, 43) and abolished by omission of extracellular Ca2+ or blockade of voltage-dependent Ca2+ channels (10, 13).

We recently reported that the endoplasmic reticulum (ER) takes up Ca2+ during the upstroke of the [Ca2+]c rise induced by an imposed depolarization and releases it slowly upon repolarization (12). Because this slow release prolongs the period of [Ca2+]c elevation, we hypothesized that it might prevent [Ca2+]c from returning to basal levels when oscillations occur at a high frequency and thus underlie the sustained elevation of [Ca2+]c on top of which the oscillations appear. The aim of the present study was thus to evaluate the possible contribution of the ER to the beta -cell [Ca2+]c response to glucose. Thapsigargin (TG) was used to inhibit the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) pump (41) and prevent the uptake of Ca2+ by the ER. However, Ca2+ emptying of the ER by TG causes sustained membrane depolarization and stable elevation of [Ca2+]c in glucose-stimulated beta -cells (6, 12, 33, 45). The contribution of the ER to [Ca2+]c oscillations cannot be studied under these conditions. Therefore, [Ca2+]c oscillations similar to those produced by glucose were imposed by repetitive depolarizations of the plasma membrane with elevated concentrations of K+ in the presence of diazoxide, an opener of K+-ATP channels. The results demonstrate that the ER markedly dampens the amplitude of [Ca2+]c oscillations and contributes to maintain a steady-state elevation of [Ca2+]c when [Ca2+]c oscillations occur at a frequency (2-3/min) similar to that of the fast oscillations produced by glucose.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation of islets and islet cell clusters. The research project was approved by, and the experiments were conducted in accordance with, the guidelines of the Commission d'Ethique d'Expérimentation Animale of the University of Louvain School of Medicine. Islets were aseptically isolated after collagenase digestion of the pancreas of fed female NMRI mice (25-30 g) killed by decapitation. When needed, they were dispersed into clusters of islet cells as previously described (25). Islets or clusters were cultured for 1-3 days in RPMI 1640 culture medium (GIBCO, Paisley, UK) containing 10% heat-inactivated fetal calf serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 10 mM glucose. Except for the experiments of Figs. 1A, 6A, and some control tests performed with overnight cultured islets, most experiments were carried out with islets or cells cultured for 2-3 days. This culture period was necessary for the tissue to firmly attach to glass coverslips and sustain frequent solution changes at a high flow rate used subsequently during [Ca2+]c measurements. Suction with glass micropipettes proved unsatisfactory to hold the islets motionless. However, the few successful experiments performed with this technique showed that the results were not different between islets cultured overnight or for 2-3 days.


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Fig. 1.   Determination of the K+ concentrations that best mimic the glucose-induced changes of membrane potential of beta -cells within islets. A: pancreatic beta -cells within single islets were first identified by their typical electrical activity during perifusion with a medium containing 10 mM glucose (G10) and a standard concentration of 4.8 mM K+. Thereafter, diazoxide (Dz) was added to the medium, and the K+ concentration was changed stepwise, as indicated. Trace is representative of results obtained with 14 islets perifused with various combinations of K+ concentrations. B: relationship between the membrane potential of pancreatic beta -cells and the logarithm of the external K+ concentration ([K+]o) in the presence of 250 µM diazoxide and 10 mM glucose. Data corresponding to the experimental curve () were obtained in experiments similar to that in A. The membrane potential in the presence of each K+ concentration was measured after stabilization at a new level. Values are means ± SE. The theoretical line () represents changes of membrane potential calculated from the Nernst equation for a K+-selective membrane and an intracellular K+ concentration of 120 mM. This K+ concentration is close to that extrapolated from the reversal potential after fitting of the experimental curve to a linear fit (dotted line).

Solutions and drugs. The medium used for the isolation of islets and for all experiments was a bicarbonate-buffered solution containing (in mM) 120 NaCl, 4.8 KCl, 2.5 CaCl2, 1.2 MgCl2, and 24 NaHCO3. It was gassed with O2-CO2 (94:6) to maintain pH 7.4 at 37°C. Except for electrophysiological experiments, it was supplemented with 1 mg/ml BSA (fraction V, Boehringer Mannheim, Mannheim, Germany). When the concentration of KCl was increased, that of NaCl was decreased accordingly to keep the osmolarity of the medium unchanged. TG was obtained from Sigma (St. Louis, MO) and diazoxide from Schering-Plough Avondale (Rathdrum, Ireland).

[Ca2+]c measurements. Cultured islets or clusters of islet cells were loaded with, respectively, 2 µM fura PE3-AM (Teflabs, Austin, TX) for 90-120 min or 1 µM fura 2-AM (Molecular Probes, Eugene, OR) for 40 min at 37°C in a bicarbonate-buffered solution containing 10 mM glucose. When needed, 1 µM TG was added to the loading solution. The coverslip was then used as the bottom of a 200-µl temperature-controlled perifusion chamber (Intracell, Royston, Herts, UK) mounted on the stage of an inverted microscope. The flow rate was 2 ml/min, and the temperature within the chamber was 37°C. The different solutions were introduced into the chamber through separate quartz capillaries placed just in front of the studied islet or cluster and were rapidly changed by computer-controlled Iso-Latch valves (Parker Hannifin, Fairfield, NY). Control experiments with two solutions containing different concentrations of K+ showed that as little as 0.2 s was enough to start changing [Ca2+]c in beta -cells with this system. [Ca2+]c was measured at 10 Hz by dual-wavelength (340 and 380 nm) excitation spectrofluorimetry, using a photometric-based system (Photon Technologies International, Princeton, NJ) to capture the emitted fluorescence at 510 nm. [Ca2+]c was calculated by comparing the ratio of the 510-nm signals successively acquired at 340 and 380 nm with a calibration curve based on the equation of Grynkiewicz et al. (18) and was established by filling the chamber with an intracellular type of solution containing 10 µM fura PE3 or fura 2 free acid, and ~10 mM or <1 nM free Ca2+. The dissociation constants, or Kd, for the fura PE3-Ca2+ and fura 2-Ca2+ complexes of, respectively, 290 (44) and 224 nM (18) were used. Previous experiments have shown that the Ca2+ probe is not significantly compartmentalized in islet cells and that the changes in fluorescence report changes in [Ca2+] within the cytosol, not the ER (12).

Electrophysiology. The membrane potential of a single beta -cell within an islet was continuously recorded with a high-resistance (~200-MOmega ) intracellular microelectrode (32). beta -Cells were identified by the typical electrical activity that they display in the presence of 10 mM glucose.

Presentation of the results. The experiments are illustrated by traces (means ± SE) or by recordings that are representative of results obtained with the indicated number of islets from at least three different preparations. The statistical significance between means was assessed by paired or unpaired Student's t-test as appropriate. Differences were considered significant at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Design of the experimental conditions to test the effect of TG. Because TG depolarizes beta -cells and usually abolishes the oscillations of membrane potential and [Ca2+]c induced by glucose (6, 12, 33, 45), the drug cannot be used to evaluate directly the contribution of the ER to glucose-induced [Ca2+]c oscillations. Therefore, all experiments were performed in the presence of 250 µM diazoxide, which, by opening K+-ATP channels (42), clamps the plasma membrane at the resting potential and prevents the depolarizing effect of TG (12, 33). Because diazoxide also prevents the depolarizing effect of glucose, islet cells were cyclically depolarized and repolarized to potentials close to those of oscillations of the membrane potential induced by glucose in the absence of diazoxide. The K+ concentrations that best mimicked these potential changes were determined by recording the beta -cell membrane potential with intracellular microelectrodes. After initial recordings in the presence of 10 mM glucose alone, diazoxide was added, and the K+ concentration of the medium was changed stepwise (4.8, 8, 10, 12, 30, and 45 mM). Each step caused a depolarization of the beta -cell membrane (Fig. 1A). At concentrations of K+ >=  10 mM, the membrane potential varied linearly with the logarithm of the K+ concentration. The slope of the best linear fit for this relationship was 59 mV for a 10-fold change of the K+ concentration, which is close to the theoretical value of 61.5 mV (at 37°C) given by the Nernst equation (Fig. 1B). This value is higher than that of 47 mV measured in experiments performed in 0-2.8 mM glucose (32), probably because diazoxide opens K+-ATP channels more efficiently than the simple omission of glucose. Below 10 mM K+, the slope decreased, indicating that the membrane potential of beta -cells is not exclusively determined by the K+ permeability.

The resting potential in the presence of 4.8 mM K+, 10 mM glucose, and diazoxide averaged -71 ± 4 mV (n = 3). The concentration of 10 mM K+ was selected to bring the membrane potential to a similar value (-61 ± 3 mV, n = 6) to that measured during the repolarization intervals between glucose-induced membrane potential oscillations without diazoxide (-62 ± 3 mV, n = 8) (Fig. 1A). The concentration of 30 mM K+ (in the presence of diazoxide) was selected to depolarize the membrane to a potential (-32 ± 2 mV, n = 13) between the peak and the foot of the spikes occurring on the plateau of membrane potential oscillations (Fig. 1A). In the presence of diazoxide, TG did not affect the absolute values or the kinetics of the changes in membrane potential induced by switches between low- and high-K+ solutions (12).

Role of the ER on [Ca2+]c in conditions mimicking a change of the glucose concentration from 3 to 10 mM. Figure 2A shows [Ca2+]c changes occurring in a mouse islet stimulated by a rise in the glucose concentration from 3 to 10 and then 15 mM. A small initial drop was followed by a large increase, and eventually by rapid oscillations occurring above a sustained elevation. These [Ca2+]c changes are known to result from concomitant changes in membrane potential (13, 38, 45).


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Fig. 2.   Contribution of the endoplasmic reticulum to cytosolic Ca2+ concentration ([Ca2+]c) changes occurring in pancreatic islets under conditions mimicking glucose-induced [Ca2+]c oscillations. A: spontaneous [Ca2+]c changes induced in a control mouse islet by stepwise increases of the glucose (G) concentration in the medium. This recording is representative of results obtained in 4 different islets. B and C: mouse islets were perifused with a medium containing 250 µM Dz throughout. Glucose and K+ concentrations (in mM) in the perifusion medium were changed as indicated. Protocol of depolarization used to induce imposed [Ca2+]c oscillations is shown (B, top). After 1-min perifusion with 4.8 mM K+, islets were depolarized with 30 mM K+ for 1 min. They were then submitted to 30 or 20 cycles of 8/16 s or 12/12 s of depolarization/repolarization with 30 and 10 mM K+, respectively. The frequency of the imposed oscillations was thus kept constant (2.5/min). After 30 min, islets were perifused with 10 mM K+ until the end. Changes in pulse protocol at 1, 14, and 22 min coincided with the indicated change in glucose concentration. B: [Ca2+]c changes in control islets; C: [Ca2+]c changes in islets pretreated with 1 µM thapsigargin (TG) during the period of loading with fura PE-3. Traces are means of results obtained in 4 (B) and 5 (C) islets.

To mimic the changes in beta -cell membrane potential induced by 10 mM glucose, the islets were submitted, in the presence of 250 µM diazoxide, to cycles of depolarization-repolarization, following a protocol that is illustrated in Fig. 2B (top). When glucose was increased from 3 to 10 mM, the islets were depolarized for 1 min with 30 mM K+ to reproduce the initial phase. Thereafter, the concentration of K+ was repetitively changed between 30 mM (for 8 s) and 10 mM (for 16 s) to impose oscillations of the membrane potential of similar duration and frequency (2.5/min) to those induced by 10 mM glucose itself under control conditions (16, 21). At the end of the experiment, the islets were continuously perifused with 10 mM K+.

In control islets, the depolarization protocol induced [Ca2+]c changes similar to those elicited by glucose in the absence of diazoxide, with a first [Ca2+]c peak followed by [Ca2+]c oscillations occurring above a sustained elevation (Fig. 2B). During the pulses of K+30/K+10 (8/16 s), [Ca2+]c oscillations had an average amplitude of 186 ± 23 nM, with a nadir at 173 ± 21 nM, i.e., 118 ± 19 nM above basal [Ca2+]c in the presence of 4.8 mM K+ (Table 1, line 1). In TG-pretreated islets, basal [Ca2+]c at 4.8 mM K+ was not modified (Table 1, lines 1 and 2), whereas both initial [Ca2+]c peak and [Ca2+]c oscillations induced by 30 mM K+ were about two times larger than in control islets (Fig. 2, B and C; Table 1, lines 1 and 2). Moreover, the nadir of [Ca2+]c oscillations during the pulses of K+30/K+10 (8/16 s) was at 95 ± 10 nM, similar to the average [Ca2+]c (107 ± 14 nM) during final repolarization with 10 mM K+, and much closer to basal [Ca2+]c in 4.8 mM K+ (Delta ) than in control islets (Table 1, compare line 2 with line 1).

                              
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Table 1.   Effects of thapsigargin on [Ca2+]c changes induced by various protocols of depolarization

Mouse islets contain ~20% non-beta -cells (25) that are preferentially located at the periphery of the organ and might thus contribute to the recorded [Ca2+]c signal. To ascertain that the results obtained in islets with the imposed depolarizations correctly reflect changes in beta -cells, the same protocol was repeated with clusters of ~10 islet cells, a preparation that contains a low proportion of non-beta -cells (9% in all clusters, with 58% of clusters entirely composed of beta -cells) (25). The depolarizing protocol induced similar qualitative results as in islets (Fig. 3 and Table 1, lines 5 and 6). Again, TG was without effect on steady-state [Ca2+]c during continuous perifusion with 4.8 and 10 mM K+, but it affected [Ca2+]c oscillations during K+ pulses. The amplitude of [Ca2+]c oscillations was increased from 371 ± 34 to 921 ± 13 nM, and the nadir was lowered from 276 ± 23 to 142 ± 11 nM, i.e., to a similar average [Ca2+]c (115 ± 9 nM) to that during prolonged repolarization with 10 mM K+ (Table 1, line 6).


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Fig. 3.   Contribution of the endoplasmic reticulum to [Ca2+]c changes occurring in clusters of islet cells during imposed depolarizations of different durations. Clusters of ~10 mouse islet cells were perifused with a medium containing 250 µM Dz throughout. Protocol was similar to that in Fig. 2, except that the glucose (G) concentration of the medium was kept at 10 mM during application of the depolarizing pulses. A: [Ca2+]c changes in control clusters. B: [Ca2+]c changes in clusters pretreated with 1 µM TG during the period of loading with fura 2. Each trace is the mean of results obtained in 3 clusters.

All of these observations indicate that the sustained [Ca2+]c elevation (Delta ) above which [Ca2+]c oscillations occur in untreated beta -cells is dependent on a functional ER. They are fully compatible with our suggestion that Ca2+ is released from the ER at the end of each oscillation (12).

Role of the ER on [Ca2+]c in conditions mimicking a change of the glucose concentration from 10 to 15 mM. Membrane potential recordings of beta -cells within an islet have shown that raising the glucose concentration of the medium from 10 to 15 mM under control conditions increases the duration of the depolarization phase without affecting the frequency of membrane potential oscillations (21). Figure 2A illustrates [Ca2+]c changes in an islet in response to a similar rise of the glucose concentration. The peak of [Ca2+]c oscillations was increased, and the nadir between the oscillations was slightly shifted upward. To mimic these changes in islets treated with diazoxide, the frequency of K+ pulses was not changed, but the duration of the depolarizing phase (K+30) was increased while that of the repolarizing phase (K+10) was decreased, both to 12 s, in accordance with the measurements of the membrane potential of islets perifused with 15 mM glucose (21). The glucose concentration was increased from 10 to 15 mM simultaneously with the change in pulse protocol. Neither in control nor in TG-pretreated islets did this protocol affect the amplitude of [Ca2+]c oscillations (Table 1, lines 1 and 2). However, the nadir between the oscillations became more elevated, the difference being larger (P < 0.05) in the absence of TG (33 ± 7 nM , n = 4) than in its presence (12 ± 4 nM, n = 5). Similar qualitative results were obtained in clusters of islet cells. Thus the rise of the nadir brought about by the change of the protocol was larger in control than in TG-pretreated cells (88 ± 14 nM, n = 4 vs. 14 ± 7 nM, respectively, n = 4, P < 0.01; Table 1, lines 5 and 6). This highlights again the role of the ER in the sustained elevation of [Ca2+]c above which the oscillations of [Ca2+]c occur in control beta -cells.

Two series of experiments were then performed to test which of the two parameters, the change of the glucose concentration or the change in the pulse protocol, affected more the nadir of [Ca2+]c oscillations. When the glucose concentration was increased from 10 to 15 mM while the pulse protocol remained unchanged (8 s of K+30 and 16 s of K+10), the nadir and the amplitude of imposed [Ca2+]c oscillations did not change either in control or TG-pretreated islets (3 experiments, data not shown). In the other series of experiments, the glucose concentration of the medium was kept constant at 10 mM, but the pulse protocol (K+30/K+10) was changed from 8/16 s to 12/12 s (Table 1, lines 3 and 4). This resulted in a significant upward shift of the average nadir, which again was larger in control than in TG-pretreated islets (39 ± 7 nM, n = 3 vs. 5 ± 2 nM, n = 3, respectively, P < 0.05; Table 1, compare lines 3 and 4). Similar results were obtained in clusters of islet cells, as shown by the mean traces presented in Fig. 3. Thus TG pretreatment virtually suppressed the upward shift of the nadir provoked by the increase of the depolarizing phase (86 ± 11 nM in control cells, n = 3 vs. 8 ± 4 nM in TG-pretreated cells, n = 3, P < 0.01; Table 1, lines 7 and 8).

When the period of depolarization becomes longer, Ca2+ influx is larger, and more Ca2+ is taken up by the ER. The larger shift of the nadir could thus reflect release of this additional Ca2+ by the ER. To test this possibility, the pulse protocol was kept constant, but Ca2+ influx was augmented by increasing stepwise the Ca2+ concentration of the medium from 0.5 to 10 mM. In control islets, the amplitude of [Ca2+]c oscillations increased with the external Ca2+ concentration (Fig. 4A), and the nadir of the oscillations became more elevated (see beginning and end of recordings in Fig. 4A). In TG-pretreated islets, the amplitude of [Ca2+]c oscillations also increased, but the nadir did not change (Fig. 4B). This shows that the contribution of the ER to the upward shift of the nadir of [Ca2+]c oscillations increases with Ca2+ influx in control beta -cells.


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Fig. 4.   Contribution of the endoplasmic reticulum to the upward shift of the nadir of [Ca2+]c oscillations increases with Ca2+ influx in islets. Mouse islets were perifused with a medium containing 250 µM Dz and 10 mM glucose (G10) throughout. The Ca2+ concentration (in mM) of the perifusion medium was changed as indicated. Protocol of depolarization used to impose [Ca2+]c oscillations is shown (A, top). After 2 min of perifusion with 10 mM K+, islets were submitted to 70 cycles of 8-s depolarization and 16-s repolarization with 30 and 10 mM K+, respectively. After 30 min, islets were perifused with 10 mM K+ until the end. A: [Ca2+]c changes in control islets; B: [Ca2+]c changes in islets pretreated with 1 µM TG during period of loading with fura PE-3. Each trace is the mean of results obtained in 3 islets.

The role of the ER in the setting of the nadir was studied further by increasing gradually the duration of the depolarization phase of each K+ pulse while keeping the frequency constant at 2.5/min (Fig. 5). The nadir gradually moved to higher [Ca2+]c in control islets (Fig. 5, A and C) but remained much more stable in TG-pretreated islets, a move to higher [Ca2+]c occurring only when the period of depolarization (30 mM K+) exceeded 75% of each depolarization-repolarization cycle (Fig. 5, B and C).


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Fig. 5.   Gradual contribution of the endoplasmic reticulum to [Ca2+]c changes occurring in islets during imposed depolarizations of different durations. Mouse islets were perifused with a medium containing 250 µM Dz and 10 mM glucose (G10) throughout. Protocol of depolarization used to impose [Ca2+]c oscillations is shown (A, top). After 2 min of perifusion with 10 mM K+, islets were submitted to cycles of depolarization and repolarization with 30 and 10 mM K+, respectively. After 58 min, they were perifused with 30 mM K+ until the end. The duration of the depolarization and repolarization phases was changed by steps of 3 s after each series of 20 cycles, as shown by the expanded pulses. The frequency of the imposed oscillations was thus kept constant (2.5/min). A: [Ca2+]c changes in control islets; B: [Ca2+]c changes in islets pretreated with 1 µM TG during the period of loading with fura PE-3. Each trace is representative of results obtained in 4 (A) and 5 (B) islets. C: average [Ca2+]c at the nadir between the oscillations; D: average amplitude of [Ca2+]c oscillations measured during the last 5 min of each pulse protocol and plotted as a function of the percentage of the time of depolarization with 30 mM K+ (means ± SE). * P < 0.05 and ** P < 0.01 for the difference between control islets and islets pretreated with TG (Thapsi). Data in C and D were calculated for all experiments like those shown in A and B.

These experiments also illustrate the major buffering capacity of the ER. The amplitude of [Ca2+]c oscillations was consistently much larger in TG-pretreated than in control islets (Fig. 5D). Interestingly, this buffering role was detectable only during the imposed oscillations. Thus steady-state elevation of [Ca2+]c during continuous depolarization with 30 mM K+ (right-hand part of Fig. 5B) was similar in control and TG-pretreated islets (378 ± 20 nM, n = 4 vs. 421 ± 38 nM, n = 5). The experiment illustrated by Fig. 5B also shows that [Ca2+]c can be lowered from ~600 to almost 100 nM within 10 s, even in the absence of a functional ER. This demonstrates that the mechanisms of Ca2+ extrusion are also very efficient in beta -cells. The progressive decrease in the peak of [Ca2+]c oscillations observed in TG-pretreated islets (Fig. 5B) but not in control islets (Fig. 5A) might be attributed to inactivation of voltage-dependent Ca2+ channels by high [Ca2+]c.

Role of the ER on [Ca2+]c during mixed [Ca2+]c oscillations. During continuous stimulation with 10 mM glucose, the [Ca2+]c response of many islets displays a mixed pattern characterized by slow oscillations with superimposed fast ones of variable frequencies (Fig. 6A). Trains of five pulses of K+30/K+10, at different frequencies and durations, were applied to the islets following the protocol shown above Fig. 6B. In control islets, this protocol of depolarization produced mixed [Ca2+]c oscillations similar to those occurring in spontaneously oscillating islets (compare mean traces of Fig. 6, A and B). Importantly, peak [Ca2+]c increased with the repetition of the rapid pulses, and the recovery of a basal [Ca2+]c at the end of the last pulse was slow. The pattern of the mixed oscillations was very different in TG-pretreated islets (Fig. 6C). The largest [Ca2+]c peak coincided with the first pulse of each train, and [Ca2+]c quickly returned to basal levels after the last pulse. Moreover, the fast oscillations occurred on nearly basal levels when the frequency of the oscillations was 2.5/min (first half of Fig. 6C). This shows that the sustained [Ca2+]c phase that slowly develops during mixed [Ca2+]c oscillations in control islets also involves the ER.


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Fig. 6.   The endoplasmic reticulum contributes to the pattern of mixed [Ca2+]c oscillations in islets. A: spontaneous mixed [Ca2+]c oscillations induced by 10 mM glucose (G10) in a mouse islet. This recording is representative of results obtained in 15 different islets. B and C: mouse islets were perifused with a medium containing 250 µM Dz and 10 mM glucose throughout. The protocol of depolarization used to impose [Ca2+]c oscillations is shown (B, top). After 2 min of perifusion with 10 mM K+, islets were sequentially submitted to 6 series of 5 cycles of depolarization and repolarization with 30 and 10 mM K+, respectively. Between each series, islets were perifused with 10 mM K+. Duration of depolarization and repolarization phases (in s) is indicated above each representative pulse. The frequency of the imposed oscillations was either 2.5/min (first 3 series of pulses) or 5/min (last 3 series of pulses). B: [Ca2+]c changes in control islets; C: [Ca2+]c changes in islets pretreated with 1 µM TG during the period of loading with fura PE-3. Each trace is the mean of results obtained in 3 (B) and 4 (C) islets.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study demonstrates that the ER limits the amplitude of [Ca2+]c oscillations and keeps [Ca2+]c elevated between oscillations when these are induced by repetitive Ca2+ influx through voltage-dependent Ca2+ channels in pancreatic beta -cells.

A summation of [Ca2+]c transients has been reported in patch-clamped single beta -cells submitted to short depolarization steps applied at a high frequency (>= 30/min) to mimic rapid action potentials (2, 37). Here, the concentration of extracellular K+ was repetitively changed at a frequency of 2.5/min to mimic the oscillations of the membrane potential occurring in beta -cells within islets during stimulation with glucose. This resulted in [Ca2+]c oscillations whose nadir remained well above basal [Ca2+]c. In other words, the imposed [Ca2+]c oscillations appeared on top of a sustained elevation of [Ca2+]c, as do glucose-induced [Ca2+]c oscillations. Suppression of this elevation by TG identifies the ER as a major contributor to its development.

We have previously suggested that periodic influx of Ca2+ into beta -cells induces parallel oscillations of the Ca2+ concentration in both cytosol and ER (12). Oscillations of the Ca2+ concentration in the ER are attributed to Ca2+ uptake during the upstroke of [Ca2+]c oscillations, followed by Ca2+ release when Ca2+ influx stops. The sustained [Ca2+]c elevation above which [Ca2+]c oscillations occur would result from differences in the kinetics of uptake and release of Ca2+ by the ER, the uptake being much faster than the release (12). The fast kinetics of the uptake by the ER can be inferred from the observation that inactivation of the SERCA by TG nearly doubled the amplitude of the [Ca2+]c rise produced by short depolarizations (3 s). The mechanism of the slow release process has not been identified, but Ca2+- or inositol phosphate-induced Ca2+ release, two fast processes, are not involved (12). We suggest that it reflects slow leakage of Ca2+ from the ER. Although such a mechanism may seem surprising, it is fully compatible with numerous observations showing that blockade of the SERCA pump by TG in a Ca2+-free medium induces a transient rise in [Ca2+]c by emptying of the ER (28, 33-35). Because of its slow kinetics, this passive Ca2+ release from the ER prolongs [Ca2+]c oscillations when influx of Ca2+ has stopped and prevents [Ca2+]c from returning to basal levels during fast [Ca2+]c oscillations like those induced by glucose.

An important contribution of this study was to show that the unexplained mixed [Ca2+]c oscillations observed in islets (23) and clusters of beta -cells (25, 36) stimulated by glucose can be reproduced by trains of depolarizations interrupted by longer pauses. It is therefore tempting to ascribe them to the mixed oscillations of the membrane potential that occur in some beta -cells during glucose stimulation (8, 24, 36), and to the activity of the ER that permits summation of these [Ca2+]c signals. This does not imply that summation of fast oscillations is the only mechanism to generate slow oscillations. Indeed, slow oscillations can occur in the absence of fast oscillations, probably as a result of long bursts of action potentials (13). The mechanisms that we propose to explain the mixed pattern of [Ca2+]c oscillations are thus different from those suggested by Liu et al. (29). From studies using ob/ob mouse islets, these authors also emphasize the role of depolarization-induced Ca2+ influx, but they attribute the mixed pattern of slow and superimposed fast oscillations to separate cell populations, each showing one type of response. They also invoke spiking Ca2+ release from the ER in some cells to coordinate the fast [Ca2+]c oscillations. We do not believe that this proposal is applicable to islets from normal mice for three reasons. First, [Ca2+]c transients resulting from Ca2+ mobilization in response to glucose and cAMP-producing agents are much more common in beta -cells from these leptin-deficient ob/ob mice than in normal mice (1). Second, experiments using two intracellular microelectrodes in the same islet have shown that the electrical activity is similar and synchronized in all beta -cells from normal mice (11, 31). Third, mixed [Ca2+]c oscillations have been observed in clusters of 2-20 cells (25, 36) and even in single beta -cells (26) from normal mice.

A rise in the glucose concentration lengthens the depolarized, active phase and shortens the repolarized, silent phase of membrane potential oscillations without affecting their frequency (21). It also causes an upward shift of the nadir between [Ca2+]c oscillations (i.e., prevents [Ca2+]c from intermittently returning to basal levels) (14, 38). A similar shift occurs when islets are subjected to a depolarizing protocol that mimics the changes in membrane potential normally produced by raising glucose from 10 to 15 mM. This elevation of the nadir cannot be explained by too short a repolarization for extrusion of all Ca2+ from the cytosol after Ca2+ influx has stopped, or by insufficient repolarization for abolition of Ca2+ influx. Thus, in TG-pretreated islets, short periods of repolarizations (9 s) are usually sufficient for restoration of low [Ca2+]c during the nadir, although the kinetics of the changes in membrane potential induced by high and low K+ solutions are not different from those in control islets (12).

When the external Ca2+ concentration was raised stepwise without changing the pulse protocol, the nadir between [Ca2+]c oscillations was progressively shifted to higher values in control but not in TG-pretreated islets. Our interpretation is that the ER takes up more Ca2+ when Ca2+ influx is larger at higher extracellular Ca2+ and therefore releases more Ca2+ during the repolarization intervals. This is consistent with the observations that high K+ raises the Ca2+ concentration in the ER of insulin-secreting rat insulinoma cells (30) and ob/ob beta -cells (40), and that the filling state of the ER is directly proportional to the steady-state [Ca2+]c level (7, 9, 12, 34). We suggest that the same mechanism explains the upward shift of the nadir observed when the fraction of depolarization time increases, for instance when the glucose concentration of the medium is raised from 10 to 15 mM. Under these conditions, Ca2+ pumping into the ER probably results from a direct activation of SERCA by Ca2+. An increase in cell metabolism is unlikely to be involved, because the change in glucose concentration (10 to 15 mM) did not affect the nadir of [Ca2+]c oscillations when the pulse protocol was kept constant. This is not surprising, because glucose-induced filling of the ER is nearly maximal at 10 mM (40).

The strong buffering capacity of the ER is illustrated by the much smaller amplitude of [Ca2+]c oscillations in control than in TG-pretreated islets. Because voltage-dependent Ca2+ currents are not affected by TG (12), this difference cannot be ascribed to greater Ca2+ influx. Whereas the buffering properties of the ER are evident during intermittent Ca2+ influx and [Ca2+]c oscillations, they are not during sustained Ca2+ influx, probably because the Ca2+ concentration within the ER is then in equilibrium with a steady-state [Ca2+]c ensured by extrusion mechanisms in the plasma membrane.

In conclusion, our study demonstrates that the ER has a major role in the control of [Ca2+]c oscillations induced by repetitive Ca2+ influx through voltage-dependent Ca2+ channels. The ER limits the amplitude of [Ca2+]c oscillations by a fast uptake and a slow release of the ion. This slow release keeps the nadir of regular [Ca2+]c oscillations elevated (higher than basal [Ca2+]c) or gradually sums the Ca2+ signals to result in mixed [Ca2+]c oscillations. This buffering effect of the ER with cycles of Ca2+ uptake and release could serve several functions. Prolongation of [Ca2+]c elevation between oscillations might recruit insulin secretory granules into a pool from which they can be released once [Ca2+]c is high enough to promote exocytosis, e.g., during the next [Ca2+]c oscillation (17). Pumping of Ca2+ into the ER could prevent [Ca2+]c from reaching too high levels that might be cytotoxic (27) and refill the organelle in which Ca2+ affects processes such as protein synthesis or maturation. Thus the proteolytic processing of proinsulin within the ER involves specific Ca2+-dependent maturation steps, and depletion of intracellular Ca2+ pools impairs proinsulin conversion and intracellular transport (19). The Ca2+ content of the ER might also modulate cell survival, because depletion of ER Ca2+ stores triggers apoptosis (46). Finally, because the Ca2+ content of the ER (45) influences the membrane potential, the periodic emptying and refilling of the ER during [Ca2+]c oscillations may participate in feedback mechanisms controlling membrane potential oscillations (12).


    ACKNOWLEDGEMENTS

This work was supported by Grant 3.4552.98 from the Fonds de la Recherche Scientifique Médicale (Brussels), Grant 1.5.212.00 from the Fonds National de la Recherche Scientifique (Brussels), Grant ARC 00/05-260 from the General Direction of Scientific Research of the French Community of Belgium, and by the Interuniversity Poles of Attraction Program (P4/21), Federal Office for Scientific, Technical and Cultural Affairs. P. Gilon is Maître de Recherches of the Fonds National de la Recherche Scientifique, Brussels.


    FOOTNOTES

Address for reprint requests and other correspondence: P. Gilon, Unité d'Endocrinologie et Métabolisme, Univ. of Louvain Faculty of Medicine, UCL 55.30, Ave. Hippocrate 55, B-1200 Brussels, Belgium (E-mail: gilon{at}endo.ucl.ac.be).

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.

First published January 2, 2002;10.1152/ajpendo.00347.2001

Received 1 August 2001; accepted in final form 18 December 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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