Voltage-dependent entry and generation of slow
Ca2+ oscillations in
glucose-stimulated pancreatic
-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 |
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
-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
-cells situated in pancreatic islets.
islet
-cells; calcium ion entry; strontium ion; cytoplasmic
calcium ion oscillations
 |
INTRODUCTION |
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 |
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.
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
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 +
× F380. The isocoefficient
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.
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Statistical analysis. Results are
presented as means ± SE. Statistical significances were evaluated
by Student's t-test for paired data.
 |
RESULTS |
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).

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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.
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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).
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-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 -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.
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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).
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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.

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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.
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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.
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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.

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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.
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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.
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 |
DISCUSSIONS |
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.
 |
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.
 |
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Ashcroft, F. M.,
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