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J Biol Chem, Vol. 274, Issue 52, 36883-36890, December 24, 1999
From the Department of Medical Cell Biology, Uppsala University,
SE-751 23 Uppsala, Sweden
Free Ca2+ was measured in
organelles of individual mouse pancreatic beta cells loaded with the
low affinity indicator furaptra. After removal of cytoplasmic indicator
by controlled digitonin permeabilization the organelle Ca2+
was located essentially in the endoplasmic reticulum (ER), >90% being
sensitive to inhibition of sarco(endo)plasmic reticulum Ca2+-ATPases. The Ca2+ accumulation in the ER
of intact beta cells depended in a hyperbolic fashion on the glucose
concentration with half-maximal and maximal filling at 5.5 and >20
mM, respectively. Also elevation of cytoplasmic Ca2+ by K+ depolarization significantly
enhanced the Ca2+ accumulation. In permeabilized beta cells
1-3 mM ATP caused rapid Ca2+ filling of the ER
reaching almost 500 µM. At 50 nM,
Ca2+ ER became half-maximally filled at 45 µM
ATP, whereas only 3.5 µM ATP was required at 200 nM Ca2+. Inositol 1,4,5-trisphosphate induced a
rapid release of about 65% of the ER Ca2+, and its
precursor phosphatidylinositol 4,5-bisphosphate was found to slowly
mobilize 75% by another mechanism. It is concluded that glucose is an
efficient stimulator of Ca2+ uptake in the ER of pancreatic
beta cells both by increasing ATP and cytoplasmic Ca2+.
Because physiological concentrations of cytoplasmic ATP are in the
mM range, Ca2+ sequestration can be anticipated
to be modulated by factors reducing its ATP sensitivity.
Ca2+ is a key regulator of many cellular functions
including the release of insulin from pancreatic beta cells (1, 2). After the introduction of membrane-permeable fluorescent indicators a
number of studies have provided important information about cytoplasmic
Ca2+ in the beta cell (3). In contrast, relatively little
is known about Ca2+ in the organelles. The present
knowledge is based essentially on in situ labeling with
45Ca (4, 5) and on measurements of the Ca2+
concentration in medium containing isolated organelles (6) or
preparations of permeabilized islets (7), islet cells (8, 9), or
tumor-derived beta cells (9-12). A problem when interpreting these
data is that the preparations include cells other than normal beta
cells and that the permeabilization is difficult to control in islets
and suspensions of cells. An alternative to these techniques is to
measure organelle Ca2+ directly. Maechler et al.
(13) recently performed such measurements in clonal INS-1 cells
specifically expressing the Ca2+-sensitive photoprotein
aequorin in the endoplasmic reticulum (ER).1 It is possible to
study organelle Ca2+ in normal cells with fluorescent
indicators introduced as membrane-permeable acetoxymethyl esters
(14-16). Taking advantage of the latter approach we recently reported
that the low affinity indicator furaptra can be used to monitor
organelle Ca2+ in individual pancreatic beta cells after
controlled permeabilization of the plasma membrane (17).
Glucose, the major natural stimulator of insulin release, has profound
effects on the beta cell handling of Ca2+ (18, 19). Early
studies with 45Ca indicated that glucose, in addition to
stimulating the entry of Ca2+ into the beta cells, promotes
the sequestration of the ion in an inositol 1,4,5-trisphosphate-
(IP3) sensitive pool (18). Because Ca2+
accumulation in the ER is required both for Ca2+
homeostasis of the beta cell (20-22) and the proinsulin handling (23),
it is important to explore whether sequestration of this ion is
actively regulated by ATP produced during glucose metabolism (2) or
simply reflects the elevation of cytoplasmic Ca2+ (13).
Using the furaptra approach we now demonstrate that glucose is an
efficient stimulator of Ca2+ uptake in the ER of the
pancreatic beta cell both by increasing ATP and cytoplasmic
Ca2+.
Materials--
Reagents of analytical grade and deionized water
were used. The acetoxymethyl ester of the Ca2+ indicator
furaptra, the heavy metal chelator
N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), thapsigargin, and IP3 were purchased from Molecular
Probes (Eugene, OR); and the acetoxymethyl ester of the
Ca2+ indicator Fura-2FF was from TefLabs (Austin, TX).
Roche Molecular Biochemicals provided collagenase, HEPES, and ATP, and
Calbiochem supplied digitonin. Phosphatidylinositol 4,5-bisphosphate
(PIP2) sodium salt solution in chloroform:methanol:water
(20:9:1) was from Lipid Products (Redhill, UK). The Ca2+
chelators EGTA, N-(2-hydroxyethyl)ethylenediaminetriacetic
acid, and nitrilotriacetic acid as well as oligomycin were obtained from Sigma. Diazoxide and heparin were kind gifts from Schering (Kenilworth, NJ) and Løvens kemiske fabrik (Ballerup, Denmark), respectively. Unless otherwise stated, intact cells were exposed to a
medium containing: 125 mM NaCl, 5.9 mM KCl, 1.2 mM MgCl2, 1.3 mM CaCl2,
and 25 mM HEPES with the pH adjusted to 7.40 with NaOH.
Permeabilized cells were superfused with an "intracellular" medium
containing: 140 mM KCl, 0-10 mM
Na2ATP, and 10 mM HEPES with the pH adjusted to
7.00 with KOH. During changes of the ATP concentration free
Mg2+ was maintained at 0.1 mM. Free
Ca2+ was buffered to 50 nM Preparation of Pancreatic Beta Cells--
Islets of Langerhans
were isolated from the splenic part of the pancreas of adult
ob/ob mice taken from a non-inbred colony (25).
Single cells were prepared by shaking the islets in a Ca2+-deficient medium (26). After suspension in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 30 µg/ml gentamicin, the cells were allowed to attach to circular cover glasses during culture for 1-5
days at 37 °C in an atmosphere of 5% CO2 in humidified
air. The ob/ob mouse islets contain more than
90% beta cells (25). Non-beta cells were avoided by selecting cells
with large size and low nuclear/cytoplasmic ratio for analyses
(27).
Loading with Ca2+ Indicator and
Permeabilization--
In most experiments the cells were loaded with
furaptra during a 60-min incubation at 37 °C with 4 µM
of its acetoxymethyl ester added to the extracellular medium containing
3 mM glucose. In some experiments the cells were instead
loaded with Fura-2FF using an analogous protocol. After rinsing, the
cover glasses with the attached cells were used as exchangeable bottoms
of an open superfusion chamber thermostated at 37 °C. The plasma
membrane was permeabilized in intracellular medium supplemented with 4 µM digitonin during simultaneous measurements of the
fluorescence obtained by excitation at 340 and 380 nm (17). The
detergent was immediately removed when there was a sudden drop in
fluorescence caused by the loss of cytoplasmic furaptra. The effect of
glucose on organelle Ca2+ was tested by 0-20
mM of the sugar in the loading medium together with 400 µM diazoxide, which hyperpolarizes the beta cells by activating the ATP-dependent K+
(KATP) channels (28). In this case the cells
were rinsed with indicator-free extracellular medium containing 50 nM Ca2+ and the same concentrations of glucose
and diazoxide. Subsequent permeabilization was performed with
maintenance of the glucose concentration in intracellular medium
containing 4 µM digitonin. Investigating the influence of
elevated cytoplasmic Ca2+, loading was performed with the
beta cells depolarized with 30.9 mM K+ in the
presence of 400 µM diazoxide and 3 mM
glucose, followed by rinsing in the same medium lacking indicator. The
permeabilization with 4 µM digitonin was made in
intracellular medium containing the same concentration of glucose and 1 µM Ca2+. Intracellular medium containing 50 nM Ca2+ but lacking digitonin and glucose was
introduced immediately upon permeabilization.
Measurements of Ca2+ in Intracellular
Stores--
Organelle free Ca2+ was measured with a dual
wavelength microfluorometric system (Deltascan, Photon Technology
International Inc, Princeton, NJ). The excitation light was alternately
directed to two monochromators by a chopper mirror spinning at 50 Hz.
The monochromator outputs were connected via a bifurcated optical fiber
to the epifluorescence attachment of an inverted microscope (Nikon
Diaphot) equipped with a 100x objective (numerical aperture 1.3).
Fluorescence was recorded at 535 nm with a photomultiplier using a
25-nm half-bandwidth interference filter. The background-subtracted signals, obtained by excitation at 340 and 380 nm, were recorded at 2 Hz using the FeliXTM software (Photon Technology International).
Digital Imaging Microscopy--
The distribution of furaptra and
Ca2+ among subcellular compartments was assessed by imaging
single beta cells at 510 nm with an intensified CCD camera (Extended
ISIS-M, Photonic Science, Robertsbridge, UK). A 75-watt xenon arc lamp
and 10-13-nm half-bandwidth interference filters were used for
excitation at 340 and 380 nm. The excitation filter changer was part of
a Magiscan image analysis system (VisiTech International, Sunderland,
UK). Images, consisting of 64 averaged video frames, were captured
every 3.1 s using the Tardis program (VisiTech International). All
images were corrected for background before calculation of the 340/380
nm fluorescence excitation ratios.
Methodological Considerations--
Furaptra has been reported to
have KD values for Ca2+ and
Mg2+ binding corresponding to 53 µM and 1.5 mM, respectively (29). These affinities are convenient for
monitoring cytoplasmic Mg2+, and such studies have been
performed also in pancreatic beta cells (30). Using furaptra for
measurements of Ca2+ in beta cell organelles, it is
therefore important to consider interference from Mg2+.
Although we have previously demonstrated that variations in Mg2+ do not affect the measurements (17), it cannot be
excluded that there is a small background contribution of this ion.
Because the KD values for furaptra depend strongly
on the experimental conditions, the concentrations of organelle
Ca2+ are presented as the 340/380 nm fluorescence
excitation ratios. To get a rough estimate of the actual organelle
concentration of Ca2+ we performed titrations by exposing
the permeabilized beta cell to increasing concentrations of the ion in
the presence of the equilibrating ionophore Br-A23187. Fig. 1
illustrates the permeabilization procedure and a subsequent
Ca2+ titration using the digital imaging technique. Beta
cells loaded with furaptra exhibited a diffuse
Ca2+-independent cytoplasmic fluorescence when excited at
340 nm with the brightest signal from the nucleus (Fig. 1A).
After permeabilization with 4 µM digitonin in the
presence of 200 nM Ca2+ and 3 mM
ATP there was a marked loss of the 340-nm excitation signal from the
cytoplasm and nucleus with a significant amount of fluorescence
remaining in cellular organelles (Fig. 1B). In the
permeabilized cell the furaptra 340/380-nm fluorescence excitation ratio displayed regional differences (Fig. 1C). The highest
ratios corresponded to a Ca2+ concentration of almost 500 µM, as indicated from Ca2+ titration in the
presence of the equilibrating ionophore Br-A23187 (Fig.
1). It is evident that the furaptra is
not Ca2+ saturated even in the compartments with the
highest concentrations of the ion.
In a medium containing Br-A23187 the presence of 3 mM
Mn2+ resulted in quenching of 94 ± 2%
(n = 5) of the fluorescence excited at 340 nm (not
shown), indicating almost complete hydrolysis of the indicator ester.
In the absence of Mn2+ there was no evidence for heavy
metal interference with the measurements. At a concentration of 20 µM the membrane-permeable heavy metal chelator TPEN
lacked detectable effects on the 340/380-nm fluorescence excitation
ratio (Fig. 2). Higher concentrations of
TPEN (200-1200 µM) induced a
concentration-dependent linear reduction of the fluorescence excitation ratio (r = 0.99;
p < 0.001; n = 4), consistent with its
low affinity binding of Ca2+ (31).
Although the control experiments with furaptra indicated that
measurements of organelle Ca2+ could be performed without
interference from Mg2+ or heavy metal ions, we also tested
whether the more Ca2+-specific indicator Fura-2FF could be
used. Unfortunately, Fura-2FF loaded poorly with little fluorescence
remaining after digitonin permeabilization. This problem was aggravated
by substantial leakage of the indicator. Titration experiments, like
those in Fig. 1, indicated that Fura-2FF in organelles reacted to
Ca2+ concentrations as low as 100 nM and became
almost saturated at 50 µM of the ion (not shown). Because
Fura-2FF was inferior to furaptra for measurements of Ca2+
in beta cell organelles, subsequent measurements were performed with
the latter indicator.
It has been reported that IP3-induced release of
Ca2+ in permeabilized hepatocytes loaded with Fura-2FF is
followed by a rapid mitochondrial uptake of some of this
Ca2+, affecting the kinetics of the release process (32).
However, these authors did not observe such an effect with
Ca2+-buffered medium or maximally effective concentrations
of IP3, as used in the present study. It is therefore
unlikely that mitochondria affect Ca2+ release from the ER
in our experiments. We have previously observed that the steady-state
organelle Ca2+ detected by furaptra in permeabilized beta
cells is influenced little by agents affecting mitochondrial function
(17). The Ca2+ concentration in the mitochondrial matrix
has been reported to be much lower in insulin-releasing cells (33, 34)
than the average Ca2+ concentration now found in the
furaptra-containing organelles. Accordingly, mitochondrial furaptra can
be expected to have a minimal influence on the observed variations in
Ca2+ signal.
Presentation of Data and Statistical Analysis--
Results are
expressed as mean values ± S.E. Differences were statistically
evaluated by a two-tailed Student's t test. The SigmaStat
software (SPSS Inc., Chicago, IL) was used for regression analyses.
Exponential curve fits and illustrations were made with the Igor Pro
software (Wavemetrics Inc., Lake Oswego, OR).
To investigate the effect of glucose on organelle
Ca2+, intact beta cells were loaded with furaptra in medium
containing 0-20 mM of the sugar under hyperpolarizing
conditions (exposure to 400 µM diazoxide). When the cells
were permeabilized in an ATP-free medium containing 50 nM
Ca2+, there was a prompt increase of the furaptra
fluorescence ratio followed by gradual decay. This decay, reflecting
emptying of the organelle pool, started from a level strongly dependent
on the glucose concentration. Fig. 3
shows experiments with beta cells maintained in medium lacking
(A) or containing 7 (B) and 20 (C)
mM glucose. In the absence of glucose there was only a tiny
increase of the furaptra fluorescence ratio upon permeabilization, whereas in the presence of 7 and 20 mM of the sugar the
magnitudes of the increase and following decay were more pronounced.
Fitting the decay to exponential functions there was no evidence for
the involvement of more than one component. The time constant observed in the presence of 20 mM glucose was 139 ± 28 s
(n = 10). After depletion of organelle Ca2+
the introduction of 3 mM ATP resulted in a rapid refilling
with a fluorescence ratio reaching similar levels as observed upon permeabilization in the presence of 20 mM glucose
(C). Subsequent addition of the sarco(endo)plasmic reticulum
Ca2+-ATPase (SERCA) inhibitor thapsigargin or omission of
ATP (not shown) resulted in a simple exponential decay as the organelle pool was emptied. Despite variations between experiments the time constants for decay of glucose- and ATP-incorporated organelle Ca2+ did not differ within experiments, the changes being
2 ± 15 s (n = 5, thapsigargin addition) and
Testing the effect of an elevated cytoplasmic Ca2+
concentration on organelle uptake of the ion, the cells were
depolarized with 30.9 mM K+ in the presence of
400 µM diazoxide and 3 mM glucose during the furaptra loading and the subsequent rinsing. The exposure to digitonin was then made in intracellular medium containing 1 µM
Ca2+ to prevent depletion of the organelles. By lowering
Ca2+ to 50 nM immediately upon permeabilization
it became possible to study the kinetics of Ca2+
disappearance from the organelles under similar conditions as in the
glucose experiments. Fig. 5A
shows that permeabilization results in a prominent peak of the furaptra
fluorescence ratio followed by gradual decay. The peak was more
pronounced than expected from the prevailing glucose concentration (3 mM) corresponding to 75 ± 5% (n = 10, p < 0.01) of the level subsequently reached with 3 mM ATP. Also in this case the early decay followed similar monoexponetial kinetics as observed when omitting ATP at the end of the
experiments. Like the effect of glucose on organelle uptake of
Ca2+, that of K+ depolarization was prevented
by oligomycin (Fig. 5B).
Glucose Regulation of Free Ca2+ in the Endoplasmic
Reticulum of Mouse Pancreatic Beta Cells*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
500
µM with 2 mM EGTA,
N-(2-hydroxyethyl)ethylenediaminetriacetic acid, or
nitrilotriacetic acid, as indicated in the figure legends. The ion
concentrations were calculated using the Maxchelator program (24). To
prepare PIP2 solutions the solvent was first evaporated by
flushing with N2. Medium was then added, and
PIP2 was dispersed by a 30-min sonication on ice. To
prevent contamination of the superfusion system (see below),
PIP2-containing solutions were added directly to the
experimental chamber.
View larger version (33K):
[in a new window]
Fig. 1.
Titration of the organelle Ca2+
concentration in an individual beta cell loaded with furaptra.
Images of the Ca2+-independent fluorescence excited at 340 nm were captured at 510 nm before (A) and after
(B) permeabilization with 4 µM digitonin in a
medium containing 3 mM ATP and 200 nM
Ca2+. Comparing A and B, it should be
noted that the gain of the image intensifier was increased before
capturing images of the permeabilized cell. After permeabilization the
340/380-nm fluorescence excitation ratio was recorded from the entire
cell (C). ATP was subsequently removed, and 2 µM of the Ca2+ ionophore Br-A23187 was added.
The Ca2+ concentration of the medium was then varied
between 0 and 10 mM using EGTA (0) or nitrilotriacetic acid
(50-500 µM Ca2+) as Ca2+ buffers
(D-J). D shows a plot of the ratio from the
region with the highest initial ratio. Representative of six
experiments.
View larger version (10K):
[in a new window]
Fig. 2.
Effect of TPEN on the organelle
Ca2+ concentration. The furaptra-loaded cells were
permeabilized with 4 µM digitonin in medium containing 3 mM ATP and 200 nM Ca2+ buffered
with EGTA. Increasing concentrations of TPEN (20-1200
µM) were then introduced. The fluorescence excitation
ratios are expressed relative to the initial values obtained in the
absence of TPEN. The solid line shows a fit of 24 individual
data points to a linear function (r = 0.990, p < 0.001). Data are mean values ± S.E. of four
experiments.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 ± 15 s (n = 5, ATP omission) between the
first and second decay. Oligomycin (2 µg/ml) completely prevented the
effect of glucose on organelle Ca2+ without interfering
with the action of ATP after permeabilization (not shown). The glucose
dependence of the Ca2+ accumulation in the beta cell
organelles is summarized in Fig. 4. The
filling observed immediately upon permeabilization after pre-exposure
to different glucose concentrations is expressed in percent of the
uptake subsequently reached with 3 mM ATP. Subtracting the
glucose-independent accumulation, the data fitted a hyperbolic function
(r = 0.905, p < 0.001) with
half-maximal effect at 5.5 mM. In accordance with the
organelle Ca2+ pool being filled during exposure to 20 mM glucose, subsequent beta cell permeabilization in the
presence of 3 mM ATP resulted in an instant increase of the
furaptra fluorescence ratio with no further change (not shown).
View larger version (17K):
[in a new window]
Fig. 3.
Effect of glucose concentration on organelle
Ca2+ in individual beta cells. The cells were loaded
with furaptra in the absence (A) or presence of 7 (B) and 20 (C) mM glucose in a medium
containing 400 µM diazoxide. After rinsing in a similar
medium lacking indicator and containing only 50 nM
Ca2+ (EGTA), the cells were exposed to 4 µM
digitonin (DIG) in an ATP-free intracellular medium with
maintenance of the Ca2+ and glucose concentrations.
Digitonin was withdrawn immediately upon permeabilization. C
shows the design of the experiments with subsequent additions of 3 mM ATP and 200 nM thapsigargin (TH).
Each trace is representative of six experiments.
View larger version (9K):
[in a new window]
Fig. 4.
Dependence of organelle Ca2+ on
glucose concentration. The filling of the organelle
Ca2+ pool immediately upon permeabilization is expressed as
the percent of the filling reached after the subsequent addition of 3 mM ATP, as illustrated in Fig. 2, and plotted as a function
of the glucose concentration. The solid line shows a fit of
31 individual data points to a hyperbolic function plus a constant
representing glucose-independent uptake (r = 0.905, p < 0.001). Data are mean values ± S.E. for six
to seven experiments.
View larger version (19K):
[in a new window]
Fig. 5.
Effect of depolarization on organelle
Ca2+ in individual beta cells. The cells were loaded
with furaptra in the absence (A) or presence (B)
of 2 µg/ml oligomycin in a medium containing 400 µM
diazoxide, 30.9 mM K+, and 3 mM
glucose. After rinsing in a similar medium lacking indicator, the cells
were exposed to 4 µM digitonin (DIG) in an
ATP-free intracellular medium containing 1 µM
Ca2+ (buffered with EGTA) with maintenance of the glucose
concentration. Digitonin and glucose were withdrawn, and
Ca2+ was lowered to 50 nM immediately upon
permeabilization. ATP (3 mM) was then introduced as
indicated. The traces are representative for 10 (A) and 5 (B) experiments.
The ATP sensitivity of the uptake process was studied in a medium
containing 50 or 200 nM Ca2+. After initial
depletion of organelle Ca2+ in an ATP-free medium,
introduction of 1 µM-1 mM ATP resulted in
concentration-dependent reuptake of the ion. In the
presence of 50 nM Ca2+ more than 10 µM ATP was required for uptake of Ca2+ (Fig.
6A), whereas there was a
considerable uptake at 1 µM ATP with 200 nM
Ca2+ (Fig. 6B). Nevertheless, the maximal
concentrations of organelle Ca2+ reached with 1 mM ATP were similar at 50 and 200 nM
Ca2+, corresponding to 2.21 ± 0.32 (n = 6) and 2.56 ± 0.34 (n = 9) ratio units,
respectively. Further elevation of ATP to 10 mM resulted in
a small decrease of the fluorescence excitation ratio at both Ca2+ concentrations.
|
In the next series of experiments the effect of Ca2+ was
tested, keeping ATP constant at 3 µM or 3 mM.
In the presence of 3 µM ATP, there was little uptake of
Ca2+ at 50 nM of the ion (Fig.
7A). Further elevations of
Ca2+ to 200 nM-5 µM resulted in
concentration-dependent increases of organelle
Ca2+. During exposure to 3 mM ATP there was a
pronounced increase of organelle Ca2+ already at 50 nM Ca2+ with no further effect during gradual
elevations of Ca2+ to 5 µM (Fig.
7B). The effects of Ca2+ and ATP on the
organelle Ca2+ stores are summarized in Fig.
8. Half-maximal uptake of organelle Ca2+ was reached at concentrations of ATP estimated to 45 and 3.5 µM at 50 and 200 nM Ca2+,
respectively (Fig. 8A). Whereas half-maximal uptake of
Ca2+ was obtained at less than 50 nM in the
presence of 3 mM ATP, about 230 nM
Ca2+ was required at 3 µM ATP (Fig.
8B). When tested with 50 nM Ca2+ in
the absence and presence of half-saturating concentrations of ATP (45 µM), 2-5 mM glucose 6-phosphate had no
effect on organelle Ca2+ (not shown).
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), nine (
), six (
), and eight
(
) experiments.
Most of the organelle Ca2+ in beta cells permeabilized
during exposure to 3 mM ATP and 200 nM
Ca2+ was released when the endoplasmic reticulum
Ca2+-ATPase was inhibited with thapsigargin (Fig.
9A). During subsequent exposure to the Ca2+ ionophore Br-A23187 the additional
release was less than 10%. The thapsigargin-sensitive Ca2+
could be divided into pools sensitive or not to IP3 (Fig.
9B). Maximally effective concentrations of IP3
(10 µM) thus released 64 ± 4% (n = 7) of the Ca2+ sensitive to thapsigargin. The fraction of
IP3-sensitive Ca2+ was somewhat lower when the
Ca2+ stores had been refilled after permeabilization during
Ca2+-depleting conditions (ATP- or
Ca2+-deficient medium). Fig.
10 illustrates such experiments,
testing the effect of IP3 at different filling states of
the thapsigargin-sensitive store. In the presence of 200 nM
Ca2+ and 3 µM ATP the depleted stores
refilled to 47 ± 7% (n = 14, Fig. 10). In this
situation IP3 released 44 ± 4% (n = 14) of the thapsigargin-sensitive Ca2+. After subsequent
maximal filling of the stores by combining 200 nM
Ca2+ with 3 mM ATP (Fig. 10A) or 1 µM Ca2+ with 3 µM ATP (Fig.
10B) the IP3-sensitive fractions did not change significantly (38 ± 4%, n = 9 and 54 ± 8%, n = 5, respectively).
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It was recently reported that PIP2 decreases the ATP
sensitivity of the KATP channels (35, 36). We
therefore investigated whether PIP2 has similar effects on
the ATP sensitivity of the organelle Ca2+ uptake. Most of
these experiments were performed at 50 nM Ca2+
to approximate the situation in unstimulated beta cells. In the presence of 45 µM ATP, which half-maximally fills the
Ca2+ stores (Fig. 8), 5 µM PIP2
was found to release 75 ± 3% (n = 6) of the
thapsigargin-sensitive Ca2+ with no additional effect of 10 µM IP3 (Fig.
11A). This release (time
constant 331 ± 46 s, n = 18) was
significantly slower than that observed after omission of ATP
(p < 0.02). Control experiments revealed that the
absence of an IP3 effect was because of depletion of the
stores rather than to PIP2 interference with the
IP3 receptors (not shown). When the
IP3-sensitive pool was first emptied, introduction of
PIP2 caused additional release of Ca2+ (Fig.
11B). Apart from releasing more Ca2+ at a slower
rate, the effect of PIP2 differed from that of
IP3 in being resistant to heparin inhibition (Fig.
12). The PIP2 effect could
not be attributed to a diminished ATP sensitivity of the Ca2+ uptake. The ATP dependence of the Ca2+
sequestration was almost identical in the presence and absence of 5 µM PIP2 (Fig.
13). Also when tested in the presence
of 200 nM Ca2+ the ATP sensitivity of the
organelle Ca2+ uptake remained unaffected by
PIP2 (not shown).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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In the present study we have investigated the glucose dependence of Ca2+ accumulation into organelles of intact beta cells and how these stores are affected by ATP, Ca2+, and polyphosphoinositides after permeabilization of the plasma membrane. After loading with the low affinity Ca2+ indicator furaptra, individual cells were subject to controlled permeabilization with a low concentration of digitonin (17). The exposure to the detergent resulted in the release of cytoplasmic furaptra leaving approximately 20% of the dye trapped in intracellular organelles. The remaining furaptra monitored a Ca2+ pool more than 90% of which was sensitive to SERCA inhibition. This Ca2+ pool was influenced little by agents affecting mitochondrial function but effectively mobilized by IP3 (17). Although IP3 may affect different cellular compartments, ER is undoubtedly most important (37). Therefore, the SERCA-dependent pool will subsequently be referred to as ER.
Glucose is the major natural stimulator of insulin release. The
increase in ATP/ADP ratio, obtained with the metabolism of the sugar,
closes the KATP channels resulting in
depolarization with elevation of cytoplasmic Ca2+ and
activation of exocytosis (38). Glucose is also an important regulator
of Ca2+ fluxes in beta cell organelles (2). Studying the
role of the sugar in ER accumulation of Ca2+, it is
important to discriminate between the effects of elevation of ATP and
of cytoplasmic Ca2+. It was recently reported that glucose
stimulates the ER accumulation of Ca2+ in clonal INS-1
cells (13). Although basal ATP was required, this sequestration was
essentially attributed to the elevation of the cytoplasmic
Ca2+ concentration. Analyzing the role of glucose as an
energy source for the ER uptake of Ca2+, we have used
diazoxide to clamp the plasma membrane close to the equilibrium
potential for K+ (28). At physiological K+
concentrations, diazoxide hyperpolarizes the beta cells, and cytoplasmic Ca2+ remains at resting concentrations when the
glucose concentration is varied in the 0-20 mM range (39).
In contrast to the observations in the INS-1 cells, we now find that
glucose potently stimulates the ER sequestration of Ca2+ in
mouse beta cells also when there is no elevation of cytoplasmic Ca2+. However, a rise of cytoplasmic Ca2+ was
also stimulatory. In the presence of 3 mM glucose the ER accumulation of Ca2+ was almost doubled by K+
depolarization. Although the latter effect can to some extent be
attributed to increased ATP production after elevation of cytoplasmic Ca2+ (40), the stimulatory effect of Ca2+ was
apparent also in the permeabilized beta cell. Our observation that the
mitochondrial ATP synthase inhibitor oligomycin abolishes ER
accumulation of Ca2+ in response to K+
depolarization or glucose exposure indicates that ATP is not only a
prerequisite for the uptake but that its production in the glycolytic
pathway is insufficient even at high concentrations of the sugar. The
crucial role of ATP was confirmed in the permeabilized beta cell, and
in accordance with studies on INS-1 (13) we did not observe that
glucose 6-phosphate regulates the Ca2+ steady state in the
ER, as reported for rat pancreatic islets (41).
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Glucose-induced elevation of cytoplasmic Ca2+ is preceded by an initial lowering (42). The opposing actions of the sugar on cytoplasmic Ca2+ involve different mechanisms. The elevation of cytoplasmic Ca2+ shows a similar sigmoidal dependence on the glucose concentration as insulin secretion (42). However, the initial lowering of cytoplasmic Ca2+ is instead hyperbolically related to the glucose concentration with a half-maximal effect at about 6 mM. We now demonstrate a similar hyperbolic dependence on glucose for the ER accumulation of Ca2+. This similarity can be taken to indicate that the glucose-induced lowering of cytoplasmic Ca2+ involves ER sequestration of the ion. Glucose has a pronounced priming action on IP3-mediated mobilization of intracellular Ca2+. The latter effect has been attributed to ER accumulation of the ion and/or increased production of the IP3 precursor PIP2 (39). The present observations indicate that ER sequestration alone can explain the priming. This conclusion is consistent with the observation that also the priming reaches half-maximum at about 6 mM glucose (39).
The uptake of Ca2+ into the ER is mediated by SERCAs. Human and rat islets of Langerhans coexpress the SERCA 2b and SERCA 3 isoforms (43), but little is known about the characteristics of Ca2+ uptake into the ER of beta cells. In the present study comparable proportions of thapsigargin-sensitive Ca2+ were mobilized by IP3 under various experimental conditions. This observation indicates that there is no major difference in uptake characteristics between the stores sensitive and resistant to IP3. In accordance with observations in other types of cells the effect of thapsigargin was fairly rapid (15, 16), indicating a pronounced leak pathway. The depletion of Ca2+ induced by SERCA inhibition mimicked that observed after the omission of ATP. It was also similar to the initial emptying of the ER Ca2+ occurring upon permeabilization of beta cells exposed to a high glucose concentration or K+ depolarization. In all cases the reduction of ER Ca2+ followed single exponential kinetics without significant differences in time constants, supporting the idea that the same pool was affected. The Ca2+ imaging data indicate that furaptra saturation cannot explain why the apparent uptake of ER Ca2+ in the presence of 3 mM ATP and 50 nM Ca2+ was not further affected by a 100-fold elevation of the Ca2+ concentration. This phenomenon may be because of the high Ca2+ buffering capacity of the ER (44). It has been demonstrated that the steady-state filling is regulated by the Ca2+ concentration in the lumen of sarco- (45) and endoplasmic reticulum (16, 46). Because of high cooperativity the Ca2+-ATPase does not generate a smoothly graded Ca2+ uptake but an on/off response (46). It is unclear why an elevation of ATP to 10 mM resulted in a decrease of the ER Ca2+. One possibility is that ATP stimulates a Ca2+ leak pathway, a phenomenon observed in other types of cells (47).
Studies on purified ER vesicles from rat islets have indicated a half-maximal Ca2+ uptake at 27 µM ATP (4). This concentration is between the 3.5 and 45 µM ATP now found to induce half-maximal uptake in permeabilized individual beta cells exposed to 200 and 50 nM Ca2+, respectively. In intact beta cells, maintained at resting concentrations of cytoplasmic Ca2+, 5.5 mM glucose induced half-maximal accumulation of ER Ca2+. A glucose regulation of the ER Ca2+ sequestration mediated by ATP is difficult to reconcile with the cytoplasmic ATP concentration, which has been estimated to be in the mM range (48, 49). However, Ca2+ uptake by the ER is not the only glucose-regulated process characterized by a paradoxically high sensitivity to ATP. It is well established that the KATP channels in pancreatic beta cells are half-maximally inhibited, when excised membrane patches are exposed to 10-15 µM ATP (38). Moreover, it was recently reported that recombinant luciferase, targeted to the cytoplasm or submembrane space of insulin-releasing MIN6 cells, exhibits a Km for ATP in the low mM range compared with µM values for purified firefly luciferase (49).
The discrepancies between the observed sensitivities to ATP in broken cell preparations and intact cells may depend on the existence of modulating cellular factors. PIP2 was recently suggested to be such a factor (35, 36), reducing the ATP sensitivity of the KATP channels sufficiently to account for physiological regulation by glucose (50). We now observe that PIP2 causes mobilization of Ca2+ from ER by a mechanism different from that of IP3. This mobilization was very slow with a rate less than half of that for the passive leakage in the presence of thapsigargin or absence of ATP. Such an effect might be expected if PIP2 acts as a partial SERCA inhibitor. However, our further experiments failed to provide evidence for any competitive effect of PIP2 shifting the ATP dependence of the Ca2+ uptake. As suggested for isolated platelet microsomes (51) and permeabilized thyroid cells (52), the slow Ca2+ release induced by PIP2 may be because of activation of a distinct mechanism. Clearly, PIP2 does not give a general explanation for the low ATP sensitivity of different cellular processes regulated by the nucleotide.
A model for glucose-induced sequestration of Ca2+ in the ER
is presented in Scheme 1. ATP provided by mitochondrial
glucose metabolism causes SERCA stimulation, explaining an early
reduction of cytoplasmic Ca2+. Subsequently ATP closes also
the KATP channels resulting in depolarization
and influx of Ca2+ through voltage-dependent
channels. The resulting elevation of cytoplasmic Ca2+ helps
to fill the ER. Both Ca2+ entering through the
voltage-dependent channels and that released from the ER by
IP3 serve to trigger exocytosis of the insulin granules. It
can be anticipated that disturbances in the generation of ATP and of
factors regulating the sensitivity to this nucleotide may contribute to
a defective release of insulin by perturbing intracellular
Ca2+ sequestration. Indeed, altered expression of SERCA
isoforms and impaired Ca2+-ATPase activity have been
described in animal models of non-insulin-dependent diabetes mellitus (43, 53, 54).
| |
ACKNOWLEDGEMENT |
|---|
We thank Aileen King for linguistic revision.
| |
FOOTNOTES |
|---|
* This work was supported by Grants 12X-562 and 12X-6240 from the Swedish Medical Research Council, the Swedish Foundation for Strategic Research, the Swedish Diabetes Association, the Novo Nordisk Foundation, Novo Nordisk Pharma AB, Family Ernfors' Foundation, Åke Wiberg's Foundation, and the Swedish Society for Medical Research.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.
To whom correspondence should be addressed: Dept. of Medical Cell
Biology, Uppsala University, Biomedicum, Box 571, SE-751 23 Uppsala,
Sweden. Tel.: 46 18 471 44 28; Fax: 46 18 471 40 59; E-mail:
erik.gylfe@medcellbiol.uu.se.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: ER, endoplasmic reticulum; IP3, inositol 1,4,5-trisphosphate; TPEN, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine; PIP2, phosphatidylinositol 4,5-bisphosphate; KATP channels, ATP-regulated K+ channels; SERCA, sarco(endo)plasmic reticulum Ca2+-ATPase.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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