From the Rolf Luft Center for Diabetes Research L6B:01, Department
of Molecular Medicine, Karolinska Institutet, Karolinska Hospital,
S-171 76 Stockholm, Sweden, the NOVO NORDISK,
BioImage, Moerkhoeg Bygade 28, 2860 Soeborg, Denmark, and the
§ Department of Medicine, University of Chicago,
Chicago, Illinois 60637
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ABSTRACT |
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In the pancreatic -cell, glucose-induced
membrane depolarization promotes opening of voltage-gated
L-type Ca2+ channels, an increase in
cytoplasmic free Ca2+ concentration
([Ca2+]i), and exocytosis of insulin. Inhibition
of Na+,K+-ATPase activity by ouabain leads to
-cell membrane depolarization and Ca2+ influx. Because
glucose-induced
-cell membrane depolarization cannot be attributed
solely to closure of ATP-regulated K+ channels, we
investigated whether glucose regulates other transport proteins, such
as the Na+,K+-ATPase. Glucose inhibited
Na+,K+-ATPase activity in single pancreatic
islets and intact
-cells. This effect was reversible and required
glucose metabolism. The inhibitory action of glucose was blocked by
pretreatment of the islets with a selective inhibitor of a
Ca2+-independent phospholipase A2. Arachidonic
acid, the hydrolytic product of this phospholipase A2, also
inhibited Na+,K+-ATPase activity. This effect,
like that of glucose, was blocked by nordihydroguaiaretic acid, a
selective inhibitor of the lipooxygenase metabolic pathway, but not by
inhibitors of the cyclooxygenase or cytochrome P450-monooxygenase
pathways. The lipooxygenase product 12(S)-HETE
(12-S-hydroxyeicosatetranoic acid) inhibited
Na+,K+-ATPase activity, and this effect, as
well as that of glucose, was blocked by bisindolylmaleimide, a specific
protein kinase C inhibitor. Moreover, glucose increased the state of
-subunit phosphorylation by a protein kinase C-dependent process.
These results demonstrate that glucose inhibits
Na+,K+-ATPase activity in Glucose metabolism increases the ATP:ADP ratio and initiates the
Further support for the proposition that glucose has effects on
membrane potential distinct from closing K+ATP
channels was provided by studies of action potential frequency in the
presence of tolbutamide and glucose (8). These studies show that
stimulatory concentrations of glucose have the ability to induce an
increased frequency of action potentials that is significantly higher
than what is observed in the presence of maximal concentrations of the
K+ATP channel blocker tolbutamide, thus
suggesting the existence of an additional regulator of The Na+,K+-ATPase is involved in maintaining
the Na+ and K+ gradients across the Materials--
Immunoprecipitation of the
Na+,K+-ATPase Isolation of Pancreatic Determination of Na+,K+-ATPase Activity
in Single Isolated Pancreatic Islets--
Single islets were isolated
and transferred individually (in 5 µl of isolation medium) to the
bottom of each well of a Nunclon-96 well plate (Nunc, Denmark). They
were kept on ice until dissection was finished (this time never
exceeded 60 min). After exposure to glucose under different protocols,
the incubations were terminated by transferring the well plate to ice.
An aliquot (50 µl) of Na+,K+-ATPase assay
medium was added: NaCl, 50 mM; KCl, 5 mM;
MgCl2, 10 mM; EGTA, 1 mM; Tris-HCl,
50 mM; Na2ATP, 10 mM (Sigma, St. Louis, MO); and [ Determination of Na+,K+-ATPase Activity
in Pancreatic Phosphorylation and Immunoprecipitation of
Na+,K+-ATPase in Intact Cells--
Determination of [Ca2+]i--
Experiments
were performed on primary cultures of Electrophysiological Studies--
Inside-out, cell-attached, and
perforated-patch configurations of the patch-clamp technique were used
(22-24). Pipettes were pulled from borosilicate glass, fire-polished,
and coated with Sylgard resin (Dow Corning) near the tips. Pipettes had
resistances between 2 and 6 M
In all experiments, the extracellular solution contained the following:
138 mM NaCl, 5.6 mM KCl, 1.2 mM
MgCl2, 2.6 mM CaCl2, and 5 mM HEPES (pH 7.4 with NaOH). For the excised-patch
configuration, the "intracellular-like" solution consisted of the
following: 125 mM KCl, 1 mM MgCl2,
10 mM EGTA, 30 mM KOH, and 5 mM
HEPES (pH 7.15 with KOH). Patches were excised into nucleotide-free solution, and ATP was first added to test for channel inhibition. ATP
was then removed, and patches were subsequently exposed to test
substances. In the cell-attached recordings, the pipette solution
consisted of the extracellular solution described above. Recordings of
membrane potential were done using the following pipette solution: 10 mM KCl, 76 mM K2SO4, 10 mM NaCl, 1 mM MgCl2, 10 mM HEPES (pH 7.35 with KOH), and 240 µg/ml amphotericin
B. All intact cell experiments (cell-attached and perforated-patch recordings) were done at 30-33 °C, whereas the inside-out patch experiments were performed at room temperature (20-22 °C). The bath
had a volume of 0.4 ml, and cells were perifused at a rate of 4 ml/min.
All test compounds were added to the perifusion medium. Each
experimental condition was tested, with identical results, in at least
five different cells.
Statistical Analysis--
Data are presented as means ± S.E. Statistical comparison of the data was performed using the paired
Student's t test or ANOVA with Sheffe's F test when
appropriate. Group differences with p < 0.05 were
considered significant.
In this study, we examined the extent to which changes in
Na+,K+-ATPase activity contribute to -cells by
activating a distinct intracellular signaling network. Inhibition of
Na+,K+-ATPase activity may thus be part of the
mechanisms whereby glucose promotes membrane depolarization, an
increase in [Ca2+]i, and thereby insulin
secretion in the pancreatic
-cell.
INTRODUCTION
Top
Abstract
Introduction
References
-cell stimulus-secretion coupling by closing ATP-regulated potassium
(K+ATP)1
channels (1). These channels are the main regulators of the
-cell resting membrane potential, and their closure will initiate membrane depolarization, opening of voltage-gated L-type
Ca2+ channels, and thereby an increase in cytoplasmic free
Ca2+ concentration ([Ca2+]i) (2-5).
However, depolarization of the plasma membrane from resting potential
to threshold potential of the voltage-dependent Ca2+ channels requires, in addition to closure of
K+ATP channels, an inward current (6). The
origin of this inward current is not known, but it has been suggested
that the current is carried by Na+ or Ca2+ (6,
7). Thus, glucose may activate an as yet unidentified component
affecting
-cell membrane potential.
-cell
membrane potential that might be affected by glucose metabolism.
-cell
plasma membrane. It extrudes three Na+ ions in exchange for
two K+ ions, generating a net outward flow of cations
through the cell membrane. This makes the pump electrogenic and results
in a hyperpolarizing effect on membrane potential. Consequently,
inhibition of Na+,K+-ATPase activity (for
example, by ouabain) leads to
-cell membrane depolarization (7, 9)
and Ca2+ influx (9, 10). It has therefore been postulated
that a decrease in Na+,K+-ATPase-mediated ion
gradients may be a contributing mechanism to insulin secretion (7-9).
The precise role of the Na+,K+-ATPase in
membrane depolarization and its possible regulation by glucose in
-cells have been difficult to define, primarily because data
reporting changes in enzyme activity have been obtained in cell
homogenates or membrane preparations, lacking intact intracellular signaling pathways. Although it was reported that glucose had an
inhibitory effect on Na+,K+-ATPase activity
(11), others failed to demonstrate significant changes in enzyme
activity (12). Nevertheless, none of the studies mentioned above have
examined short-term effects of glucose, i.e. within the time
frame matching the initial cellular events in the insulin secretory process.
EXPERIMENTAL PROCEDURES
-subunit was performed using a
polyclonal antibody raised against the whole enzyme (kindly provided by
E. Féraille, University of Geneva, Switzerland). Fura-2/acetoxymethyl ester, ouabain, mannoheptulose, arachidonic acid,
ethoxyresorufin, nordihydroguaiaretic acid, indometacin, bisindolylmaleimide, and 12,13-phorbol dibutyrate were purchased from
Sigma. Haloenol lactone
(E)-6-(bromomethylene)-3-(1-naphtalenyl)-2H-tetrahydropyran-2-one (HELSS) was obtained from Calbiochem (San Diego, CA).
12(S)-HETE, 12(R)-HETE, 20-HETE, 5-HETE, and
15-HETE were purchased from Cayman Chemicals (Ann Arbor, MI); all of
these compounds were dissolved in ethanol (final concentration,
<0.001%) except for HELSS, which was dissolved in Me2SO
(final concentration, <0.01%). AA was dissolved in ethanol under
N2 flow, and the stock solution was stored at
20 °C
protected from light. Appropriate control with the diluents was exercised.
-Cells--
All experiments were
performed in single isolated pancreatic islets and in
-cells
obtained from adult (12 months old) obese (ob/ob) mice from a local
colony (13). The mice were killed by decapitation. Pancreatic islets
were isolated by collagenase digestion. Cells were obtained by
dissociation of islets into single cells and small cell clusters (14,
15). This preparation consists of 90-95%
-cells (15). The medium
used for islet and cell isolation and [Ca2+]i
measurements contained the following: NaCl, 125 mM; KCl,
5.9 mM; CaCl2, 1.3 mM;
MgCl2, 1.3 mM; HEPES, 25 mM, pH
7.4; and 1 mg/ml bovine serum albumin. For
Na+,K+-ATPase activity determinations, bovine
serum albumin was omitted. Primary cultures of
-cells were kept in
RPMI 1640 medium supplemented with 10% fetal calf serum, 100 µg/ml
streptomycin, and 100 IU/ml penicillin. Cells were cultured on glass
coverslips or kept in suspension by gentle agitation.
-32P]ATP (NEN Life Science Products)
(specific activity 3000 Ci/mmol) in tracer amounts (1.3 nCi/µl).
Islets were transiently exposed to a thermic shock (10 min at
20 °C) in order to render the plasma membrane permeable to ATP.
The plate was sealed in order to prevent evaporation and transferred to
37 °C for additional 15 min. The incubation was terminated by
placing the well plate on ice and adding 175 µl of trichloroacetic
acid/charcoal solution. Thereafter, the content of each well was
transferred to Eppendorf tubes and centrifuged (12,000 × g for 5 min), and an aliquot from the supernatant (containing the liberated 32P) was counted in a liquid
scintillation counter. In each experiment, equivalent to one animal,
total and ouabain-insensitive Na+,K+-ATPase
activity were determined in eight islets each, and the difference
between means of these measurements represents one Na+,K+-ATPase data point.
Na+,K+-ATPase activity was expressed as nmol of
Pi/islet/h.
-Cells--
Na+,K+-ATPase
activity was measured essentially as described above (16). Freshly
isolated cells in suspension (50 µl) were incubated at room
temperature with the desired agonists. The incubation was terminated by
rapid cooling of the samples to 4 °C. Aliquots (10 µl) of the cell
suspension (protein concentration, 10-20 µg) were rapidly
transferred to the Na+,K+-ATPase assay medium
(final volume, 100 µl; see composition above). Cells were transiently
exposed to a thermic shock (10 min at
20 °C), in order to render
the plasma membrane permeable to [
-32P]ATP. The
samples were then incubated at 37 °C for 15 min. The reaction was
terminated by rapid cooling to 4 °C and addition of a mixture of
trichloroacetic acid/charcoal (5%/10%). After separating the charcoal
phase (12,000 × g for 5 min) containing the
nonhydrolyzed nucleotide, the 32P liberated in the
supernatant was counted. Na+,K+-ATPase activity
was calculated as the difference between test samples (total ATPase
activity) and samples assayed in a medium devoid of Na+ and
K+ and in the presence of 2 mM ouabain
(ouabain-insensitive ATPase activity). Protein determination was
performed according to Bradford (17), using a conventional dye reagent
(Bio-Rad, Richmond, CA).
-Cells
in suspension were labeled for 3 h at 37 °C in a buffer
containing the following: 120 mM NaCl, 5 mM
KCl, 4 mM NaHCO3, 1 mM
CaCl2, 1 mM MgSO4, 0.2 mM NaH2PO4, 0.15 mM
Na2HPO4, 5 mM glucose, 10 mM lactate, 1 mM pyruvate, 20 mM
HEPES, and 1% bovine serum albumin, pH 7.45, with the addition of 100 µCi/ml 32P-orthophosphate (NEN Life Science Products).
Incubations with either 3 or 15 mM glucose, in the presence
or absence of 1 µM bisindolylmaleimide, were performed at
room temperature. The incubations were terminated by removing the
medium and addition of cold immunoprecipitation buffer.
Immunoprecipitation of the Na+,K+-ATPase
-subunit was performed as described (18). Briefly, aliquots were
incubated overnight at 4 °C with 50 µl of rabbit polyclonal
antibody and the simultaneous addition of excess protein A-Sepharose
beads (Pharmacia Biotech, Uppsala, Sweden). Samples were analyzed by
SDS-PAGE using the Laemmli buffer system (19). Proteins were
transferred to polyvinylidene difluoride membranes (Immobilon-P,
Millipore, Bedford, MA) and subjected to autoradiography.
-cells as described before
(20, 21). Briefly,
-cells cultured for 12-24 h on glass coverslips
were incubated in basal medium (3 mM glucose) for 30-40
min with 1 µM Fura-2/acetoxymethyl ester (Sigma). After
washing, the subsequent measurements were performed in a SPEX
Fluorolog-2 CM1T11I system connected to an inverted Zeiss Axiovert 35 M epifluorescence microscope. During each experimental protocol (described in detail in the legend to Fig. 3), cells were
superfused in a custom-built chamber (20, 21). The excitation and
emission wavelengths were 340/380 and 510 nm, respectively. The results
are presented as 340/380 ratios.
. Single-channel currents
were recorded from inside-out or cell-attached membrane patches.
Channel activity in excised patches was measured at 0 mV pipette
potential (Vp), whereas single channel activity was recorded from
intact cells (cell-attached) at Vp
70 mV. Membrane potential was
monitored using the perforated-patch configuration, using amphotericin
B as pore-forming agent. Current and voltage were recorded using an
Axopatch 200 patch-clamp amplifier (Axon Instruments Inc., Foster City,
CA). During experiments, the current and voltage signals were stored
using a VR-100A digital recorder (Instrutech Corp., Elmont, NY) and a
high-resolution video cassette recorder (JVC, Tokyo, Japan). Channel
records are displayed according to the convention with upward
deflections denoting outward currents.
RESULTS
-cell
membrane depolarization and thereby to opening of L-type
voltage-dependent Ca2+ channels and influx of
Ca2+. Cells were exposed to 1 mM ouabain, a
concentration that induced maximal inhibition of the pancreatic
-cell Na+,K+-ATPase activity. The effect of
ouabain on
-cell membrane potential was determined using the
perforated-patch configuration of the patch-clamp technique. Cells were
perifused with a nonstimulatory concentration (4 mM) of
glucose. Addition of ouabain to the medium resulted in prompt (within
60-100 s) depolarization of membrane potential (Fig.
1A), which increased from a
resting potential of approximately
55 mV to a plateau potential of
about
40 mV, upon which action potentials were superimposed.
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Fig. 1.
Effect of glucose and ouabain on -cell
membrane potential and [Ca2+]i.
A, recording of membrane potential in a single
-cell
using the perforated-patch configuration of the patch-clamp technique.
The cell was perifused with a solution containing 4 mM
glucose, which resulted in a membrane potential of approximately
50
mV. Upon inclusion of 1 mM ouabain, as indicated by the
bar, the cell depolarized within 60 s to approximately
40 mV, which gave rise to a train of action potentials. B,
effect of ouabain (1 mM) on [Ca2+]i
in a Fura-2 loaded
-cell cluster. Effect of ouabain (1 mM) in the presence or absence of external
Ca2+. All protocols were terminated by addition of 15 mM glucose. The tracings are representative of four
experiments, each performed in three separate cell preparations.
C, ouabain and single K+ATP channel
activity. i, lack of effect of ouabain on channel activity
in inside-out patches. In the presence of ATP, channel activity was
almost completely blocked, but it reappeared upon removal of the
nucleotide. Addition of ouabain, as indicated by the bar,
did not affect channel activity. This lack of effect was observed in
five different cell preparations. ii, ouabain was also
without effect on K+ATP channel activity in
intact cells, as measured in the cell-attached mode. The lower tracing
was obtained 2 min after the addition of ouabain and is representative
of seven experiments from three separate cell preparations.
iii, cell-attached recording from a
-cell at 5 mM glucose exhibits a low action potential frequency.
Inclusion of ouabain in the perifusion medium dramatically increased
the frequency of action potentials. The recording was performed at
32 °C and is representative of five different cells.
The effect of ouabain on [Ca2+]i was determined in small clusters of 5-10 cells (Fig. 1B). Addition of 1 mM ouabain to cells incubated in the presence of 4 mM glucose induced a rapid and reversible increase (130 ± 17% above basal, n = 18, p < 0.001) in [Ca2+]i. A subsequent challenge with 15 mM glucose did not significantly increase [Ca2+]i further (153 ± 15% above basal, n = 18, p = 0.323 versus ouabain) when compared with the increase obtained with ouabain alone. The increase in [Ca2+]i elicited by ouabain was completely abolished in the absence of extracellular Ca2+ and rapidly restored upon addition of 1.3 mM Ca2+ to the perifusion medium. Moreover, the presence of 50 µM D-600, a voltage-dependent L-type Ca2+ channel blocker, abolished the increase in [Ca2+]i.
The Na+,K+-ATPase activity is inhibited by Ca2+ (25). Although this effect has not been reported to occur in intact cells but rather in a cell free system, the effect of glucose on Na+,K+-ATPase activity was examined after omitting Ca2+ from the extracellular medium. In the absence of Ca2+ and simultaneous presence of extracellular EGTA (0.5 mM), 10 mM glucose was still able to inhibit Na+,K+-ATPase activity (60 ± 8% of control, n = 6, p < 0.05).
Because the K+ATP channel is essential in
regulating pancreatic -cell membrane potential (5, 6), one may
question whether the depolarizing effect of ouabain is brought about
via a direct and/or an indirect effect on this channel. To examine
these possibilities, we studied K+ATP channel
activity in excised patches as well as in intact cells. As seen in Fig.
1C, ouabain had no effect on channel activity in inside-out
patches, suggesting that ouabain has no direct effect on the
K+ATP channel complex. It has recently been
reported that ouabain induces a reduction in the number of open
K+ATP channels in intact
-cells and that
this might be the mechanism whereby ouabain depolarizes the
-cell
membrane and thereby increases [Ca2+]i (10). To
correlate this finding with acute regulation of
Na+,K+-ATPase activity, we studied channel
activity using the cell-attached mode of the patch-clamp technique. To
increase the number of active channels and thereby the resolution of
channel events, we performed the experiments in the absence of glucose.
As seen in Fig. 1C, ouabain did not affect channel activity
within 2 min of exposure, the time period during which the cell
depolarizes and an increase in [Ca2+]i is
observed. Mean K+ATP channel currents were not
altered (3 ± 4%, n = 7, not significant versus control prior exposure to ouabain). Ouabain was,
however, still able to induce action potentials in the
-cell when
perifused with a threshold concentration of glucose (Fig.
1C).
We next studied the effect of glucose on Na+,K+-ATPase activity in single isolated pancreatic islets. Incubation of single medium size islets (2.4 ± 0.4 µg of protein, n = 5) with glucose resulted in a time-dependent (ANOVA p < 0.01) and dose-dependent (ANOVA p < 0.005) inhibition of Na+,K+-ATPase activity (Fig. 2, A and B). Ouabain-insensitive ATPase activity was not affected by glucose (control, 39.2 ± 2.3, n = 8 versus 15 mM glucose, 38.2 ± 3.1, (nmol of Pi/islet/h) n = 8). The inhibitory effect of glucose was reversible (Fig. 2C). After exposure to 15 mM glucose for 10 min at 37 °C, the medium (5 µl) was removed and replaced with a medium containing 3 mM glucose for additional 10 min. After this washout period, Na+,K+-ATPase activity returned to control levels.
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In order for glucose to serve as a secretagogue, it has to be
metabolized. Likewise, glucose needs to be metabolized in order to
inhibit Na+,K+-ATPase activity. The presence of
an equimolar concentration (15 mM) of the glycolytic
inhibitor mannoheptulose substantially prevented the inhibitory effect
of glucose (Fig. 2C). That glucose needs to be metabolized
and the requirement of an intact intracellular signaling system were
further supported by the lack of effect of glucose on
Na+,K+-ATPase activity in a -cell-free
system (not shown), similar to what has been previously reported
(12).
Na+,K+-ATPase activity was also determined in
-cells in suspension. Na+,K+-ATPase activity
(nmol of Pi/mg of protein/min) in this preparation was
39 ± 1, n = 20, whereas the ouabain-insensitive
ATPase was 37 ± 3 (n = 20). After isolation,
-cells were suspended in 3.6 mM glucose for
approximately 10 min at room temperature. A further increase in the
glucose concentration induced a dose-dependent decrease in
Na+,K+-ATPase catalytic activity, with a
maximal effect at 7.5 mM of glucose (ANOVA,
p < 0.01). Changes in enzyme activity were also time-dependent and occurred as early as 1 min after glucose
administration and persisted for at least 5 min (ANOVA,
p < 0.001). The ouabain-insensitive ATPase activity
was not affected by glucose (control, 37.4 ± 4, n = 4 versus 15 mM glucose, 39.0 ± 2, n = 7, not significant). Also, the effect of glucose on
Na+,K+-ATPase activity in
-cell suspension
was reversible (not shown).
The inhibitory action of glucose requires its metabolism and was absent
when a broken cell preparation was used. The pancreatic -cell is
endowed with a phospholipase A2 that is regulated in a
Ca2+-independent fashion by the increase in ATP resulting
from glucose metabolism (26, 27). This phospholipase is inhibited by
HELSS. It was previously reported that pretreatment of pancreatic
-cells with HELSS prevented the increase in
[Ca2+]i and insulin secretion induced by glucose
but not KCl or carbachol (28). Pancreatic
-cells from ob/ob mice
treated with 25 µM HELSS for 30 min at room temperature
did not change islet Na+,K+-ATPase activity
significantly, whereas HELSS abolished the inhibitory effect of glucose
(Fig. 3).
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Activation of -cell phospholipase A2 by glucose results
in hydrolysis of membrane phospholipids and the consequent increase in
the production of arachidonic acid (27). Incubation of single islets
with arachidonic acid (AA) resulted in a dose-dependent inhibition of Na+,K+-ATPase activity (Fig.
4A), with a half-maximal
inhibitory concentration of approximately 4 nM. We
evaluated the effect of glucose and AA on
Na+,K+-ATPase activity in the presence and
absence of relative selective inhibitors of the three main pathways of
AA metabolism. The cyclooxygenase pathway was inhibited with
indomethacin (Indo), the cytochrome P450-dependent
monooxygenase with ethoxyresorufin (ETX) (29), and the lipooxygenase
pathway with nordihydroguaiaretic acid (NDGA) (30). In previous
studies, incubation with each pathway inhibitor alone for as long as 30 min at room temperature did not alter Na+,K+-ATPase activity in renal proximal
tubules (31). Similarly, none of the inhibitors affected the enzyme
activity in isolated pancreatic islets (not shown). However, the
inhibitory effect of glucose (15 mM) or AA on
Na+,K+-ATPase activity (10 nM) was
completely blocked by 0.1 mM of the lipooxygenase inhibitor
NDGA (Fig. 4B). In contrast, neither ETX nor Indo modified
the inhibition of islet Na+,K+-ATPase by either
glucose or AA. Because the effect of glucose was abolished by NDGA, we
next examined whether 12(S)-HETE, the main lipooxygenase
metabolite found in pancreatic
-cells (32), inhibited
Na+,K+-ATPase activity. 12(S)-HETE
inhibited Na+,K+-ATPase activity in a
dose-dependent manner, with a half-maximal inhibitory
concentration of ~1 nM (Fig. 4C). We have also
compared the effect of 12(S)-HETE with that of other
eicosanoids on Na+,K+-ATPase activity (Table
I). Whereas 1 nM
12(S)-HETE significantly inhibited
Na+,K+-ATPase activity (as in Fig. 4) the same
concentration of the 12(R)-HETE isomer did not. 20-HETE
induced a ~20% inhibition of Na+,K+-ATPase
activity as described previously in renal epithelial cells (31). The
lipooxygenase metabolites 5- and 15-HETE did not significantly affect
Na+,K+-ATPase activity.
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It has been reported that the action of glucose on insulin secretion
involves translocation of protein kinase C (PKC) (33). In the present
study, the effect of glucose on Na+,K+-ATPase
activity was blocked by the specific PKC inhibitor Bis (Fig.
5A). Similarly, Bis blocked
the inhibitory effect of 12(S)-HETE. Additional experiments
were performed in the presence of exogenous activators of PKC. Phorbol
ester (phorbol 12,13-dibutyrate) inhibited Na+,K+-ATPase activity (vehicle, 14.6 ± 1.2, n = 5 versus 10 nM phorbol 12,13-dibutyrate, 7.1 ± 1.7, n = 5, p < 0.05), and this inhibition was not affected by
HELSS (6.6 ± 1.9, n = 5, not significant). Accordingly, in -cells labeled with 32P-orthophosphate,
glucose increased the state of phosphorylation of
Na+,K+-ATPase
-subunit, and this effect was
blocked by Bis (Fig. 5B).
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We examined the impact of PKC inhibition or 12(S)-HETE on
glucose-regulated -cell membrane potential using the perforated patch configuration of the patch clamp technique (Fig.
6). A typical recording following an
increase in the glucose concentration from 3 to 15 mM is
shown in Fig. 6A. An increase in glucose concentration leads
to depolarization of membrane potential, followed by a train of action
potentials, which ceases as the concentration of glucose is lowered to
3 mM. Preincubation of
-cells with 1 µM of
the PKC inhibitor Bis for 30 min and its continuous presence throughout the experiment did not affect glucose-induced electrical activity (Fig.
6B). Inclusion of 12(S)-HETE was without effect
on membrane potential or electrical activity at 3 or 15 mM
glucose (Fig. 6, C and E, respectively). At 6 mM glucose, which is an intermediate glucose concentration
at which the
-cell membrane potential slowly fluctuates,
12(S)-HETE was also unable to affect electrical activity (Fig. 6D).
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DISCUSSION |
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Although it has been suggested that the activity of the
Na+,K+-ATPase in the pancreatic -cell
affects electrical properties of the plasma membrane (34), its
regulation by glucose has remained controversial (11, 12). One reason
for this may be that previous investigations were performed on
broken-cell preparations in which glucose metabolism was not preserved.
In this study, we present evidence that glucose indeed causes a rapid,
reversible, and dose-dependent inhibition of the
Na+,K+-ATPase, and we suggest possible
mechanisms involved in this effect. The experiments were performed in
intact, single islets or in
-cells in suspension, under conditions
that preserve signal-transduction pathways, including those necessary
for glucose metabolism. The requirement for glucose metabolism is
supported by the lack of effect of glucose on
Na+,K+-ATPase activity in the presence of
mannoheptulose, which inhibits glucokinase (Ref. 35 and references
therein) and thereby blocks further glucose metabolism.
Several intracellular signaling messengers have been associated with
the insulin secretory process induced by glucose. Among the earliest
events in the signaling cascade is the activation of phospholipase
A2, which is Ca2+-independent and is regulated
by glucose and ATP (27). Similarly, the inhibitory effect of glucose on
Na+,K+-ATPase activity is
Ca2+-independent and is blocked by HELSS, a selective
blocker of certain PLA2 isoforms (36). Phospholipase
A2 has been localized both in the plasma membrane and in
the cytosol, but only in the latter location is it regulated by ATP
(27), suggesting that its translocation to the plasma membrane could be
a possible mechanism of activation and targeting to the specific
substrate. Despite the specificity of HELSS for the
Ca2+-independent PLA2, we cannot exclude the
contribution of other HELSS-insensitive PLA2 isoforms,
which release arachidonic acid within the -cells.
Among the PLA2, the preferred substrates are the
plasmalogens, rather than diacyl substrates (27). The enzyme catalyzes the hydrolysis of arachidonate from the sn-2 position of
choline and ethanolamine phospholipids. In the present study, AA
decreased Na+,K+-ATPase activity in a
dose-dependent manner, similar to results reported earlier
in kidney tubules (31). This observation could explain previous reports
that AA induces -cell membrane depolarization and thereby
facilitates Ca2+ entry and insulin secretion (37).
Inhibition of Na+,K+-ATPase activity by glucose
occurred independently of [Ca2+]i. Moreover,
glucose-induced AA metabolism and eicosanoid release do not require
Ca2+ influx (37).
Arachidonate 12-lipooxygenase products are involved in the secretory
process of insulin (38, 39). Pancreatic -cells (but not
-cells)
are endowed with a specific 12-lipooxygenase responsible for the
production of 12(S)-HETE (32). The 12-lipooxygenase generates 12(S)-hydroperoxyeicosatetraenoic acid
(12(S)-HPETE) from AA, and 12-HPETE is further reduced by
peroxidase to 12-HETE. This compound can also be formed by the action
of monooxygenases, but apparently not in pancreatic islets, in which
12-HETE is produced by the action of 12-lipooxygenase (40). In our
studies, blocking the 12-lipooxygenase pathway by means of the
selective inhibitor NDGA abolished the effect of both AA and glucose on
Na+,K+-ATPase activity. Furthermore, the
12-lipooxygenase product 12(S)-HETE decreased islet
Na+,K+-ATPase activity in a
dose-dependent manner. Considering that upon addition of
glucose there is an increased production of HETEs (41) and that
decreased 12-lipooxygenase activity is associated with inhibition of
insulin secretion, these results suggest that glucose is likely to
modulate Na+,K+-ATPase activity by increasing
the cellular levels of 12(S)-HETE. This hypothesis is
further supported by the findings that neither ETX nor Indo affects
islet Na+,K+-ATPase activity in response to
glucose or AA.
Receptor-mediated regulation of Na+,K+-ATPase
activity in epithelial cells is a complex phenomenon that involves the
activation and integration of diverse intracellular signals (42, 43). It appears that the signaling cascade initiated by glucose metabolism and increased cellular ATP in islet cells is followed by activation of
phospholipase A2 and subsequent stimulation of AA and
12(S)-HETE formation (Fig. 7).
The molecular links between 12(S)-HETE and the
Na+,K+-ATPase may include phosphorylation
and/or internalization of Na+,K+-ATPase
subunits, similar to what has been described in transporting epithelia
(18, 44-46). The possibility that 12(S)-HETE directly activates PKC (47-50) is supported by the fact that the inhibitory effect of glucose or 12(S)-HETE on
Na+,K+-ATPase activity was abolished by the
presence of a PKC inhibitor (Fig. 4) and that in intact, metabolically
labeled -cells, the state of phosphorylation of the
Na+,K+-ATPase
-subunit was increased by
glucose in a process that was PKC-dependent (Fig. 5). The
fact that only the PKC
and
isoforms are translocated in
response to glucose (33) may suggest that either one or both of these
isoforms are involved in the regulation of
Na+,K+-ATPase activity.
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The electrogenic nature of the Na+,K+-ATPase
has been well documented in excitable tissues (51). In pancreatic
-cells, inhibition or activation of the pump is followed by rapid
changes in membrane potential (34, 52), indicating that the pump is
electrogenic also in this tissue. Thus, as the
Na+,K+-ATPase is inhibited, it induces
depolarization of the
-cell plasma membrane (34). The present
finding that glucose induces a pronounced inhibitory effect of the
Na+,K+-ATPase suggests that part of the
depolarizing effect of glucose is due to a decrease in the
Na+,K+-ATPase-generated current. This would
allow the Na+,K+-ATPase to contribute to the
resting membrane potential, as well as to serve as a modulator of
electrical activity subsequent to glucose stimulation.
We have previously demonstrated that down-regulation of PKC activity
does not dramatically affect the effect of glucose on cytoplasmic free
Ca2+ concentration, reflecting effects on membrane
potential or insulin secretion (21). However, in those experiments, PKC
activity was down-regulated by prolonged (overnight) incubation with
phorbol esters. Because this treatment will only prevent the action of PKCs that are phorbol ester-sensitive, three isoforms out of eight, we
performed experiments in the presence of a more specific inhibitor of
PKCs, such as Bis. The present data confirm our previous observations and clearly suggest that the role of PKC in glucose-induced insulin secretion should be considered to be of a modulatory rather than of a
direct regulatory nature. In the present study, we demonstrated that
glucose-induced inhibition of the Na+,K+-ATPase
is mediated by PKC. However, it is clear that the effect of this
inhibition in -cell membrane depolarization should only be minor and
may therefore not be resolved. Similarly, the 12(S)-HETE isomer did not affect membrane potential at resting (3 mM),
threshold (6 mM), and stimulatory (15 mM)
concentrations of glucose. Hence, we believe that inhibition of
Na+,K+-ATPase activity by second messengers,
but not ouabain, is not sufficient to depolarize the plasma membrane
and that a combined action of glucose on K+-ATP channels
and Na+,K+-ATPase is needed to achieve such an
effect. This is also in agreement with the lack of effect of
12(S)-HETE on insulin secretion. Together with the
observation that PKC blockade does not affect membrane potential, these
results add further support to the hypothesis that inhibition of
Na+,K+-ATPase activity does not have a direct
regulatory effect but rather a modulatory effect on insulin release.
Our results do not support the concept that the depolarizing effect of ouabain is due to a decreased K+ATP channels activity, as previously suggested (10). It should be noted that Grapengiesser et al. (10) reported an effect on K+ATP activity following 15 min of exposure to ouabain. We were unable to demonstrate an effect of ouabain on K+ATP activity within 2 min, which is the time frame for glucose-induced inhibition of Na+,K+-ATPase activity as well as for ouabain-induced membrane depolarization and increase in [Ca2+]i (this study and Refs. 34 and 52).
In conclusion, this study presents compelling evidence for a distinct
intracellular signaling pathway activated by glucose that results in
inhibition of -cell Na+,K+-ATPase. Although
this process is initiated by activation of a Ca2+-independent phospholipase A2 and
activation of a lipooxygenase metabolite (2(S)-HETE), it is
activation of PKC and thereby increased phosphorylation of the
-subunit that is responsible for
Na+,K+-ATPase inhibition. Further studies have
to clarify how important this inhibitory effect on
Na+,K+-ATPase activity is in relation to the
overall effects of glucose in the
-cell stimulus-secretion coupling.
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ACKNOWLEDGEMENT |
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We thank Martin Wahl for useful discussions.
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FOOTNOTES |
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* This study was supported by the Swedish Medical Research Council (Grants 19X-10860, 03X-09890, 03XS-12708, and 03X-09891), funds of the Karolinska Institutet, the Swedish Diabetes Association, the Nordic Insulin Foundation Committee, the Berth von Kantzows Foundation, the Åke Wibergs Foundation, and the Emil and Vera Cornells 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. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 46-8-517-75727; Fax: 46-8-517-73658; E-mail: alejan{at}enk.ks.se.
The abbreviations used are: K+ATP, ATP-regulated K+ channels; PKC, protein kinase C; Bis, bisindolylmaleimide; HELSS, haloenol lactone (E)-6-(bromomethylene)-3-(1-naphtalenyl)-2H-tetrahydropyran-2-one; AA, arachidonic acid; Indo, indometacin; ETX, ethoxyresorufin; NDGA, nordihydroguaiaretic acid; PLA2, Ca2+ -independent phospholipase A2; ANOVA, analysis of variance; 12(S)-HETE, 12-S-hydroxyeicosatetranoic acid.
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REFERENCES |
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