Voltage-dependent entry and generation of slow Ca2+ oscillations in glucose-stimulated pancreatic beta -cells

Staffan Dryselius, Eva Grapengiesser, Bo Hellman, and Erik Gylfe

Department of Medical Cell Biology, Uppsala University, Biomedical Center, S-751 23 Uppsala, Sweden


    ABSTRACT
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Abstract
Introduction
METHODS
RESULTS
DISCUSSIONS
References

The role of voltage-dependent Ca2+ entry for glucose generation of slow oscillations of the cytoplasmic Ca2+ concentration ([Ca2+]i) was evaluated in individual mouse pancreatic beta -cells. Like depolarization with K+, a rise of the glucose concentration resulted in an enhanced influx of Mn2+, which was inhibited by nifedipine. This antagonist of L-type Ca2+ channels also blocked the slow oscillations of [Ca2+]i induced by glucose. The slow oscillations occurred in synchrony with variations in Mn2+ influx and bursts of action currents, with the elevation of [Ca2+]i being proportional to the frequency of the action currents. A similar relationship was obtained when Ca2+ was replaced with Sr2+. Occasionally, the slow [Ca2+]i oscillations were superimposed with pronounced spikes temporarily arresting the action currents. It is concluded that the glucose-induced slow oscillations of [Ca2+]i are caused by periodic depolarization with Ca2+ influx through L-type channels. Ca2+ spiking, due to intracellular mobilization, may be important for chopping the slow oscillations of [Ca2+]i into shorter ones characterizing beta -cells situated in pancreatic islets.

islet beta -cells; calcium ion entry; strontium ion; cytoplasmic calcium ion oscillations


    INTRODUCTION
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Abstract
Introduction
METHODS
RESULTS
DISCUSSIONS
References

GLUCOSE-STIMULATED INSULIN release is mediated by a rise of the cytoplasmic Ca2+ concentration ([Ca2+]i) of the pancreatic beta -cell (11, 25). The rise in [Ca2+]i is usually manifested as slow oscillations (0.1-0.5/min) arising from close to basal levels (6, 7). We proposed ten years ago that these oscillations depend on periodic depolarization of the beta -cells with resulting entry of Ca2+ through voltage-activated channels (6, 7). However, it has also been argued that the slow oscillations of [Ca2+]i observed in response to constant sugar concentrations are independent of changes in membrane potential and are mediated by Ca2+ channels insensitive to dihydropyridines (14). The latter conclusion was based on the persistence of "beta -cell-identical" slow oscillations in clonal HIT-T15 beta -cells during exposure to the L-type Ca2+ channel blocker nifedipine and on the observation that Mn2+, which was assumed to permeate only through non-voltage-dependent channels, readily enters glucose-stimulated beta -cells.

We have now evaluated the involvement of voltage-dependent Ca2+ entry for glucose generation of slow oscillations of [Ca2+]i in individual pancreatic beta -cells. It is shown that nifedipine counteracts the effects of glucose, abolishing the slow oscillations of [Ca2+]i as well as the sugar-induced influx of Mn2+. The slow [Ca2+]i rhythmicity was found to be synchronized with bursts of action currents, subject to temporary interruptions by spikes of [Ca2+]i.


    METHODS
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Abstract
Introduction
METHODS
RESULTS
DISCUSSIONS
References

Chemicals. Reagents of analytical grade and deionized water were used. Fura 2 and its acetoxymethyl ester were from Molecular Probes (Eugene, OR). HEPES, nifedipine, and bovine serum albumin (fraction V) were provided by Sigma Chemical (St. Louis, MO). Fetal calf serum was bought from GIBCO (Paisley, Scotland), and collagenase was from Boehringer Mannheim (Mannheim, Germany). Diazoxide was kindly donated by Schering-Plough (Kenilworth, NJ).

Preparation of beta -cells. Islets of Langerhans were isolated by collagenase digestion from pancreases of ob/ob mice from a local colony (10). These islets consist of >90% beta -cells, which respond normally to glucose and other regulators of insulin release (9). Free cells were prepared by shaking the islets in a Ca2+-deficient medium (15). The cells were suspended in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 30 µg/ml gentamicin and were allowed to attach to circular 25-mm coverslips during culture at 37°C in RPMI 1640 medium for 1-3 days in an atmosphere of 5% CO2.

Measurements of cytoplasmic Ca2+ and Mn2+ quench experiments. Loading of cells with the indicator fura 2 was performed during 40 min of incubation at 37°C in a HEPES-buffered medium (25 mM; pH 7.4) containing 0.5 mg/ml bovine serum albumin, 138 mM NaCl, 5.9 mM KCl, 1.2 mM MgCl2, 1.28 mM CaCl2, 3 mM glucose, and 1 µM fura 2-acetoxymethyl ester. The coverslips with attached cells were then used as exchangeable bottoms of an open 0.16-ml chamber perfused at a rate of 1 ml/min with a medium lacking indicator. Temperature control (37°C) was obtained by keeping the microscope within a climate box regulated by an air stream incubator.

The microscope was equipped with an epifluorescence illuminator and a ×100 oil immersion fluorescence objective. A rotating filter changer provided excitation light flashes at 340 and 380 nm, and the emission was measured at 510 nm using a photomultiplier. The fluorescence excitation ratio (R) was digitized at 2 Hz. The autofluorescence was negligible and not compensated for. [Ca2+]i was calculated according to Grynkiewicz et al. (8) using a dissociation constant (KD) of 224 nM and the equation
[Ca<SUP>2+</SUP>] = <IT>K</IT><SUB>D</SUB> × <FR><NU>F<SUB>0</SUB></NU><DE>F<SUB>s</SUB></DE></FR> × <FR><NU>(R − R<SUB>min</SUB>)</NU><DE>(R<SUB>max</SUB> − R)</DE></FR>
where F0 and Rmin are the fura 2 fluorescence at 380 nm and the 340-to-380 nm fluorescence excitation ratio, respectively, in an "intracellular" K+-rich medium lacking Ca2+. Fs and Rmax are the corresponding data obtained with a saturating concentration of Ca2+.

Influx through the voltage-dependent Ca2+ channels was estimated by measuring the rate of Mn2+ quenching of the fura 2 fluorescence at the Ca2+-insensitive wavelength of the dye. Instead of measuring at the isosbestic wavelength of fura 2, a Ca2+-insensitive "isosbestic" fluorescence signal (Fi) was calculated as Fi = F340 + alpha  × F380. The isocoefficient alpha  scales the negative F380 response to compensate exactly for the positive F340 response (fluorescence at 340 and 380 nm, respectively) when [Ca2+]i is increased (27). To compensate for differences in the absolute fluorescence between experiments, the Mn2+ quenching was expressed as a percentage of the basal fluorescence decrease due to influx of Mn2+ after subtracting the decrease caused by fading and leakage of fura 2. To illustrate variations in Mn2+ influx during glucose-induced [Ca2+]i oscillations, Fig. 5 also shows the rate of changes in the Ca2+-independent fluorescence. The rate was calculated with Igor Pro software (WaveMetrics, Lake Oswego, OR) after denoising with the Igor Pro implementation of the discrete wavelet transform. Among the available wavelets, Pseudo Coifman was most effective.

Parallel measurements of [Ca2+]i or cytoplasmic Sr2+ concentration and electrical activity. Loading of cells with the indicator fura 2 was performed as above using a HEPES-buffered medium (10 mM; pH 7.4) containing 142 mM NaCl, 4 mM KCl, 1.2 mM MgCl2, 2.56 mM CaCl2 or 5 mM SrCl2, 3 mM glucose, and 1 µM fura 2-acetoxymethyl ester. In this case, the open experimental chamber had a volume of 0.75 ml and was perfused at a rate of 1 ml/min with medium lacking indicator. The inverted microscope was an epifluorescence-equipped Zeiss Axiovert 100, and temperature control (32°C) was obtained by heating the chamber holder and the ×100 oil immersion fluorescence objective separately. A rotating filter changer provided excitation light flashes at 340 and 380 nm, and the emission was measured at 510 nm using a photomultiplier. The fluorescence excitation ratio was digitized at 2 Hz. [Ca2+]i was calculated as described under Measurements of cytoplasmic Ca2+ and Mn2+ quench experiments. The Sr2+ complex of fura 2 has spectral properties similar to that of Ca2+, allowing measurements at the same wavelengths (13). However, because calibration is difficult (16), the cytoplasmic Sr2+ concentration ([Sr2+]i) data were simply presented as 340/380 nm fluorescence excitation ratios. The time constants for decay of [Ca2+]i and [Sr2+]i after termination of action currents were obtained by fitting the data to single exponential functions using the Igor Pro software. Almost identical rate constants were obtained when such fits were based on calculated [Ca2+]i values or the corresponding ratio data.

The electrical activity was recorded with an EPC-9 amplifier (HEKA Electronik, Lambrecht/Pfaltz, Germany) using the cell-attached configuration of the patch-clamp technique. Currents were filtered at 2 kHz, digitized at 47 kHz (VR-10B digital data recorder; Instrutech, Great Neck, NY), and analyzed with the Igor Pro software. The current traces shown in Figs. 5 and 6 were filtered at 40 Hz. Action currents were identified after wavelet denoising. Among the available wavelets, Daubechies 8 was found to be the most appropriate for separating action currents from noise. Figure 1 illustrates the effectiveness of the procedure. The frequency of the action currents was determined by counting during overlapping 100-ms periods. The frequency data are presented after 21-point box smoothing.


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Fig. 1.   Wavelet detection of action currents. A: unfiltered recording obtained with the cell-attached configuration of the patch-clamp technique. B: same recording after denoising as described in C-E. C: time-expanded section of the original recording. D: same section after denoising with the Daubechies 8 wavelet using a 45% cutoff level. E: data from the original recording within narrow windows (broken lines) centered around the detected action currents. Data outside these windows were set to 0.

Statistical analysis. Results are presented as means ± SE. Statistical significances were evaluated by Student's t-test for paired data.


    RESULTS
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Abstract
Introduction
METHODS
RESULTS
DISCUSSIONS
References

Individual pancreatic beta -cells exposed to 3 mM glucose exhibited a low and stable [Ca2+]i in the 60-90 nM range. The depolarization obtained by increasing the medium concentration of K+ from 5.9 to 30.9 mM induced a prompt 300-600 nM elevation of [Ca2+]i. In most cases, an initial peak was followed by a decline to an intermediate plateau, which remained until the K+ concentration was reduced (Figs. 2 and 3). The Ca2+-insensitive fluorescence signal from the fura 2 indicator was not affected by K+ depolarization but decreased slowly with time due to bleaching and loss of the dye from the beta -cells. When 100-200 µM Mn2+ was added, the Ca2+-insensitive fluorescence signal declined more rapidly due to the quenching effect of Mn2+ entering the cell. K+ depolarization in the continued presence of Mn2+ promptly increased the quenching to 904 ± 73% of the initial rate (P < 0.001; n = 11 experiments) when compensating for the Mn2+-independent component (Fig. 2). Although fura 2 does not reliably detect [Ca2+]i after addition of Mn2+, it was apparent that the increased rate of quenching coincided with elevation of [Ca2+]i. Subsequent addition of 5-10 µM nifedipine immediately abolished the effect of K+, reducing the quenching to 43 ± 11% of the initial rate. There was a parallel return to basal [Ca2+]i, although this effect was partially masked by the presence of Mn2+ as revealed by separate control experiments (not shown). In other experiments, increase of the glucose concentration from 3 to 20 mM accelerated the Mn2+ quenching to 494 ± 60% of the basal rate (P < 0.001; n = 12 experiments), but the onset of this effect was delayed by 1.4 ± 0.1 min (Fig. 3, left). During the delay, there was a small lowering of [Ca2+]i, and the increased rate of quenching coincided with elevation of [Ca2+]i. Glucose failed to increase the rate of Mn2+ quenching in beta -cells hyperpolarized with 400 µM diazoxide or exposed to 5-10 µM nifedipine (Fig. 3, middle and right).


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Fig. 2.   K+ depolarization increases Mn2+ quenching of the fura 2 fluorescence in beta -cells, and this effect is inhibited by nifedipine (Nifed). Fura 2-loaded beta -cell was initially exposed to a medium containing 3 mM glucose. K+ concentration was increased from 5.9 to 30.9 mM (equimolar replacement with Na+), and 100 µM Mn2+ and 5 µM nifedipine were present as indicated. Calculated Ca2+-independent fluorescence of fura 2 is shown above cytoplasmic Ca2+ concentration ([Ca2+]i) data. Broken lines indicate additions of K+ and nifedipine. Representative of 11 experiments. In 6 experiments, 200 µM Mn2+ and 10 µM nifedipine were used. Note that the indicated levels of [Ca2+]i are not reliable after addition of Mn2+ (shaded area). Arbit units, arbitrary units.


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Fig. 3.   Glucose (Glu) increases Mn2+ quenching of the fura 2 fluorescence in beta -cells, and this effect is inhibited by nifedipine and hyperpolarization. Fura 2-loaded beta -cells were initially exposed to a medium containing 3 mM glucose. K+ concentration was increased from 5.9 to 30.9 mM (equimolar replacement of Na+) and 100 µM Mn2+, 400 µM diazoxide, 5 µM nifedipine, and 20 mM glucose were present as indicated. Calculated Ca2+-independent fluorescence of fura 2 is shown above [Ca2+]i data. Broken lines indicate times for addition of glucose and increased rate of quenching. Representative of 12, 9, and 19 experiments in left, middle, and right, respectively. In some of these experiments, 200 µM Mn2+ and 10 µM nifedipine were used. Note that indicated levels of [Ca2+]i are not reliable after addition of Mn2+ (shaded areas).

beta -Cells exposed to 11 mM glucose exhibited slow oscillations of [Ca2+]i (Fig. 4). These oscillations disappeared during exposure to 5 µM nifedipine. By using 50 µM Mn2+, it was possible to study quenching during glucose-induced [Ca2+]i oscillations (Fig. 5). To better illustrate variations in Mn2+ influx, the rate of change in Ca2+-insensitive fluorescence is also shown. It is clear that the start of the oscillations coincide with increased influx of Mn2+ (Fig. 5, line c), which peaks when the [Ca2+]i oscillations reach a plateau (line b) and decreases rapidly in parallel with [Ca2+]i during decline of the oscillations (line a).


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Fig. 4.   Nifedipine inhibits glucose-induced slow oscillations of [Ca2+]i in beta -cells. Fura 2-loaded beta -cell was initially exposed to medium containing 3 mM glucose. Before the start of the trace, slow oscillations of [Ca2+]i were induced by increasing the glucose concentration to 11 mM. Nifedipine (5 µM) was present as indicated. Representative of 3 experiments.


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Fig. 5.   Mn2+ influx is synchronous with variations in [Ca2+]i in glucose-stimulated beta -cells. Fura 2-loaded beta -cell was initially exposed to medium containing 3 mM glucose. Sugar concentration was then increased to 11 mM, and 50 µM Mn2+ was present as indicated. Calculated Ca2+-independent fluorescence of fura 2 is shown above the rate of change in this fluorescence (-dFluorescence/dt). Broken lines a-c facilitate comparison of traces. Representative of 5 experiments. Note that the indicated levels of [Ca2+]i are not reliable after addition of Mn2+ (shaded area).

When the electrical activity of beta -cells was recorded with the cell-attached configuration of the patch-clamp technique, 5 µM nifedipine was found to immediately abolish glucose-induced action currents (not shown). Attempts to simultaneously record [Ca2+]i oscillations and electrical activity proved difficult at 37°C, and the temperature was therefore reduced to 32°C. Although the cell-attached configuration of the patch-clamp technique leaves the studied cell essentially intact, repetitive slow [Ca2+]i oscillations were observed in only 5 out of >100 beta -cells. In such successful experiments, there was a close relationship between [Ca2+]i and electrical activity. Figure 6 shows an example of a single beta -cell with two subsequent [Ca2+]i oscillations paralleled by bursts of action currents. It is evident that the initial rise in [Ca2+]i coincides with the start of the action currents. During the two oscillations, there was an apparent correlation between the magnitude of the [Ca2+]i elevation and the prevailing frequency of the action currents, which increases in the beginning and decreases at the end of the bursts. After cessation of the first burst of action currents, [Ca2+]i decayed with a time constant of 21 s until the next burst started. In another cell with a distinctly terminated burst, the corresponding time constant was 19 s (not shown). A striking phenomenon in Fig. 6 is a pronounced [Ca2+]i spike during the first oscillation associated with a temporary disappearance of the action currents. This phenomenon is illustrated on an expanded time scale in Fig. 7, which also gives another example with three subsequent [Ca2+]i spikes associated with disappearance of the action currents. The action currents disappear when [Ca2+]i is close to maximal and reappear when the spikes terminate after 3-5 s.


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Fig. 6.   Glucose-induced slow oscillations of [Ca2+]i in beta -cells coincide with bursts of action currents (Curr). Fura 2-loaded beta -cell was initially exposed to medium containing 3 mM glucose. Before the start of the traces, slow oscillations of [Ca2+]i were induced by increasing the glucose concentration to 11 mM. Membrane currents and the frequency of action currents are shown above the [Ca2+]i data. Times for the start and stop of action currents are indicated by broken lines. Representative of 5 experiments.


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Fig. 7.   [Ca2+]i spiking is associated with the disappearance of action currents in beta -cells. Two fura 2-loaded beta -cells were initially exposed to a medium containing 3 mM glucose. Before the start of the traces, slow oscillations of [Ca2+]i were induced by increasing the glucose concentration to 11 mM. Membrane currents are shown above the [Ca2+]i data. Trace on left shows time expansion of the first spike in Fig. 6. Trace on right provides other examples of disappearing action currents during [Ca2+]i spiking. This phenomenon was observed in 3 cells.

With the use of 5 mM Sr2+ as a substitute for Ca2+, it was possible to demonstrate slow [Sr2+]i oscillations and action currents in parallel in four beta -cells, one of which was studied for >40 min. Figure 8 shows six subsequent oscillations and the corresponding variations in action current frequency. Although an increase in the frequency of the action currents seems to parallel the rise in [Sr2+]i, the maximal frequency was reached 22 ± 4 s before the peak elevation of [Sr2+]i. The frequency eventually decreased in parallel with [Sr2+]i, but [Sr2+]i was still elevated at the cessation of the bursts. The subsequent decay of [Sr2+]i occurred with a time constant of 50 ± 3 s. These relationships are illustrated in Fig. 9, which shows the averages of the [Sr2+]i and action current data during the six oscillations.


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Fig. 8.   Glucose-induced slow oscillations of [Sr2+]i in beta -cells coincide with bursts of action currents. Fura 2-loaded beta -cell was initially exposed to medium containing 5 mM Sr2+ and 3 mM glucose. Before the start of the traces, slow oscillations of [Sr2+]i were induced by increasing the glucose concentration to 11 mM. Frequency of action currents is shown above [Sr2+]i data. Times for the start and stop of action currents are indicated by broken lines. Cytoplasmic Sr2+ concentration ([Sr2+]i) oscillations with parallel bursts of action currents were observed in 4 cells.


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Fig. 9.   Changes in action current frequency precede variations in [Sr2+]i in glucose-stimulated beta -cells. Figure presents average action current frequency (dotted trace) and [Sr2+]i (solid trace) from 6 oscillations in Fig. 8. Broken line indicates the stop of action currents.


    DISCUSSIONS
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Abstract
Introduction
METHODS
RESULTS
DISCUSSIONS
References

Glucose stimulation of the pancreatic beta -cell is associated with an initial slow depolarization and lowering of [Ca2+]i (4). When the membrane potential reaches a threshold, opening of voltage-dependent Ca2+ channels results in action potentials and a sudden rise in [Ca2+]i. The channels can be classified as L-type, since they are activated around -50 mV and are sensitive to dihydropyridines (21). T-type Ca2+ channels are probably not involved, since they are believed to be inactivated under physiological conditions or even absent in mouse beta -cells (2). It has been proposed that non-voltage-dependent Ca2+ channels are also important for glucose-stimulated insulin release. One alternative is activation of Ca2+ or nonselective cation channels by depletion of intracellular Ca2+ stores (3, 26). However, these channels are apparently not involved in glucose generation of the slow oscillations of [Ca2+]i/[Sr2+]i. In individual beta -cells, the oscillations persist after emptying intracellular Ca2+ pools with inhibitors of the sarcoplasmic-endoplasmic reticulum Ca2+-ATPase (16). Moreover, such inhibitors promote the slow oscillatory pattern in intact pancreatic islets (18). In addition to the store-operated pathway, glucose has been reported to activate other Ca2+ channels resistant to nifedipine (22). Such channels, rather than the voltage-dependent ones, have been suggested to underlie the slow oscillations of [Ca2+]i in beta -cells exposed to constant sugar concentrations (14).

Various experimental approaches support the idea that the slow oscillations of [Ca2+]i indeed reflect periodic depolarization with opening of the L-type voltage-dependent Ca2+ channels. The glucose-induced oscillations disappear under hyperpolarizing conditions (16) and during exposure to the channel blocker methoxyverapamil (7). Moreover, it has been demonstrated with the patch-clamp technique that individual beta -cells exhibit bursts of action potentials/currents with a similar frequency as for the slow oscillations of [Ca2+]i (12, 24). Using concentrations of nifedipine, which lacked effects on glucose-induced slow oscillations of [Ca2+]i in HIT-T15 beta -cells (14), we now found that the action potentials and the slow oscillations are rapidly extinguished in mouse beta -cells. Nifedipine also blocked the influx of Mn2+ into beta -cells depolarized by glucose or excessive K+, and diazoxide-induced hyperpolarization prevented glucose from stimulating such influx. Our observations emphasize a fundamental role for voltage-dependent Ca2+ channels in the slow oscillations of [Ca2+]i and demonstrate that Mn2+, contrary to the previous assumption (14), can enter through these channels also in pancreatic beta -cells. Further studies are needed to clarify whether the failure to detect a nifedipine effect on the slow [Ca2+]i oscillations in HIT-T15 beta -cells indicates that these oscillations are different from the "apparently identical" oscillations in normal beta -cells.

Glucose-stimulated beta -cells located within the pancreatic islets exhibit repetitive bursts of action potentials (5, 20). This characteristic pattern, often referred to as slow waves, actually represents fast oscillations of the membrane potential (typically 3-5 bursts/min). Parallel measurements of electrical activity and [Ca2+]i have shown that these bursts are perfectly synchronized with fast oscillations of [Ca2+]i (23). For the first time, we now demonstrate that the 10-fold slower oscillations of [Ca2+]i in isolated beta -cells occur in synchrony with bursts of action currents and variations in Ca2+ (Mn2+) influx. Although both the fast [Ca2+]i oscillations of beta -cells situated in islets and the slow ones in isolated beta -cells coincide with bursts of action potentials/currents, there are important differences apart from the frequency. Whereas the fast islet oscillations somehow depend also on intracellular mobilization of Ca2+, the slow ones do not (18). Indeed, the fast islet rhythmicity is rapidly transformed into the slow one by agents interfering with intracellular Ca2+ mobilization.

The glucose-induced slow oscillations were characterized by a parallelism between the elevation of [Ca2+]i/[Sr2+]i and the prevailing frequency of the action currents. After the action currents terminated, [Ca2+]i decayed exponentially with a time constant of ~20 s. This rate is almost identical to the slow component of the decay in [Ca2+]i observed when glucose-stimulated beta -cells are hyperpolarized to -70 mV after a brief depolarization to 0 mV (4). Although the present study, like previous ones (16-18), indicate great similarities between the beta -cell handling of Ca2+ and Sr2+, we now found that the decay in [Sr2+]i after termination of action currents occurred at a rate only 40% of that for [Ca2+]i. This difference indicates that the removal of Sr2+ from the cytoplasm by organelle uptake and outward transport is slower than that of Ca2+.

In some experiments, there were pronounced spikes superimposed on the slow oscillations of [Ca2+]i. Previous studies have indicated that such spikes can result from depolarization-induced formation of inositol 1,4,5-trisphosphate (IP3), mobilizing Ca2+ from intracellular stores (17). The spiking is promoted by agents raising the concentration of cAMP, which may sensitize the IP3 receptors. In the absence of glucagon-producing alpha -cells or cAMP-elevating agents, such spiking is rare, now being observed in <5% of the cells. The spikes were found to coincide with temporary arrest of the action currents. A similar effect has been observed in beta -cells dialyzed with guanosine 5'-O-(3-thiotriphosphate) and has been believed to represent activation of a hyperpolarizing K+ conductance by Ca2+ released from intracellular stores (1, 19). Moreover, an analogous hyperpolarizing conductance has been reported in <5% of small beta -cell clusters stimulated with glucose (1). It was therefore suggested that intracellular Ca2+ mobilization may be involved in the hyperpolarization, which leads to termination of the rapid bursts in islets. The present study is the first demonstration that [Ca2+]i spiking causes the predicted arrest of the action currents in glucose-stimulated beta -cells. The hyperpolarizing current apparently fails to terminate the slow oscillations of [Ca2+]i in isolated beta -cells. However, based on studies of intact pancreatic islets, we recently proposed that the fast burst pattern is generated when [Ca2+]i spiking triggers hyperpolarizing currents in sufficiently many cells to make it the dominating current within a syncytium of islet cells (18). Accordingly, the characteristic pattern of fast oscillations in beta -cells situated in pancreatic islets may be due to chopping of the glucose-induced slow oscillations of [Ca2+]i into shorter ones.


    ACKNOWLEDGEMENTS

We are indebted to Matti Larsson and Aileen King for introduction into wavelets and linguistic revision, respectively.


    FOOTNOTES

This study was supported by Grants 12X-562 and 12x-6240 from the Swedish Medical Research Council and by grants from the Swedish Diabetes Association, the Swedish National Board of Health and Welfare, the Novo Nordisk Foundation, and the Family Ernfors Foundation.

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. §1734 solely to indicate this fact.

Address for correspondence and reprint requests: E. Gylfe, Dept. of Medical Cell Biology, Biomedical Centre, Box 571, S-751 23 Uppsala, Sweden (E-mail: erik.gylfe{at}medcellbiol.uu.se).

Received 14 June 1998; accepted in final form 12 November 1998.


    REFERENCES
Top
Abstract
Introduction
METHODS
RESULTS
DISCUSSIONS
References

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Am J Physiol Endocrinol Metab 276(3):E512-E518
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