INTRODUCTION

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GLUCOSE-STIMULATED INSULIN release is mediated by a rise of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) of the pancreatic -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 -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 "-cell-identical" slow oscillations in clonal HIT-T15 -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 -cells.

We have now evaluated the involvement of voltage-dependent Ca²⁺ entry for glucose generation of slow oscillations of [Ca²⁺]_i in individual pancreatic -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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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 -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% -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.

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.

View larger version (39K): [in this window] [in a new window]   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.

	RESULTS

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Individual pancreatic -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 -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 -cells hyperpolarized with 400 µM diazoxide or exposed to 5-10 µM nifedipine (Fig. 3, middle and right).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   K+ depolarization increases Mn2+ quenching of the fura 2 fluorescence in -cells, and this effect is inhibited by nifedipine (Nifed). Fura 2-loaded -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.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Glucose (Glu) increases Mn2+ quenching of the fura 2 fluorescence in -cells, and this effect is inhibited by nifedipine and hyperpolarization. Fura 2-loaded -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).

-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).

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Nifedipine inhibits glucose-induced slow oscillations of [Ca2+]i in -cells. Fura 2-loaded -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.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Mn2+ influx is synchronous with variations in [Ca2+]i in glucose-stimulated -cells. Fura 2-loaded -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 -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 -cells. In such successful experiments, there was a close relationship between [Ca²⁺]_i and electrical activity. Figure 6 shows an example of a single -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.

View larger version (26K): [in this window] [in a new window]   Fig. 6.   Glucose-induced slow oscillations of [Ca2+]i in -cells coincide with bursts of action currents (Curr). Fura 2-loaded -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.

View larger version (22K): [in this window] [in a new window]   Fig. 7.   [Ca2+]i spiking is associated with the disappearance of action currents in -cells. Two fura 2-loaded -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 -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.

View larger version (27K): [in this window] [in a new window]   Fig. 8.   Glucose-induced slow oscillations of [Sr2+]i in -cells coincide with bursts of action currents. Fura 2-loaded -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.

View larger version (18K): [in this window] [in a new window]   Fig. 9.   Changes in action current frequency precede variations in [Sr2+]i in glucose-stimulated -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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Glucose stimulation of the pancreatic -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 -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 -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 -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 -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 -cells (14), we now found that the action potentials and the slow oscillations are rapidly extinguished in mouse -cells. Nifedipine also blocked the influx of Mn²⁺ into -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 -cells. Further studies are needed to clarify whether the failure to detect a nifedipine effect on the slow [Ca²⁺]_i oscillations in HIT-T15 -cells indicates that these oscillations are different from the "apparently identical" oscillations in normal -cells.

Glucose-stimulated -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 -cells occur in synchrony with bursts of action currents and variations in Ca²⁺ (Mn²⁺) influx. Although both the fast [Ca²⁺]_i oscillations of -cells situated in islets and the slow ones in isolated -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 -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 -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 -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 -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 -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 -cells. The hyperpolarizing current apparently fails to terminate the slow oscillations of [Ca²⁺]_i in isolated -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 -cells situated in pancreatic islets may be due to chopping of the glucose-induced slow oscillations of [Ca²⁺]_i into shorter ones.