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-glucosidase activation is
Ca2+ regulated
Department of Pharmacology, University of Lund, S-223 62 Lund, Sweden
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ABSTRACT |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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An important signal involved in
glucose-stimulated insulin secretion is transduced through the action
of a lysosomal acid, glucan 1,4-
-glucosidase. We investigated the
Ca2+ dependency of this enzyme
activity in relation to insulin release. In isolated islets, increased
levels of extracellular Ca2+
induced a large increase in acid glucan 1,4--glucosidase activity accompanied by a similar increase in insulin release at both
substimulatory and stimulatory concentrations of glucose. At low
glucose the Ca2+ "inflow"
blocker nifedipine unexpectedly stimulated enzyme activity without
affecting insulin release. However, nifedipine suppressed 45Ca2+
outflow from perifused islets at low glucose and at
Ca2+ deficiency when intracellular
Ca2+ was mobilized by carbachol.
This nifedepine-induced retention of
Ca2+ was reflected in increased
acid glucan 1,4--glucosidase activity. Adding different
physiological Ca2+ concentrations
or nifedipine to islet homogenates did not increase enzyme activity.
Neither selective glucan 1,4--glucosidase inhibition nor the ensuing
suppression of glucose-induced insulin release was overcome by a
maximal Ca2+ concentration. Hence,
Ca2+-induced changes in acid
glucan 1,4--glucosidase activity were intimately coupled to similar
changes in Ca2+-glucose-induced
insulin release. Ca2+ did not
affect the enzyme itself but presumably activated either glucan
1,4--glucosidase-containing organelles or closely interconnected messengers.
pancreatic islets; lysosomal enzymes; nifedipine; emiglitate; carbachol; calcium ion
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INTRODUCTION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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IT IS A WELL-KNOWN FACT (8, 25, 34, 35) that
Ca2+ plays an essential role in
the stimulus-secretion coupling for insulin release. The cytosolic
accumulation and intracellular distribution of
Ca2+ are important for a growing
number of different events in the secretory process, which is affected
not only by the major secretagogue glucose but also by a variety of
cholinergic, adrenergic, and peptidergic influences (2, 3, 8-10,
12, 22, 25, 33-35). Moreover, glucose-induced insulin release
itself is a complex cascade of events, the details of which are far
from elucidated (2, 25, 35). We have previously proposed (14-20,
28-32) that one of these glucose-induced multiple signals is
transduced via the vacuolar system, involving the activation of a
lysosome-acid glucan 1,4--glucosidase system. The acid glucan
1,4--glucosidase (EC 3.2.1.3) and the acid -glucosidase (EC
3.2.1.20) are members of the -glucosidehydrolase family. The acid
glucan 1,4--glucosidase is known to preferentially attack
-1,4-linked polymers such as glycogen (24) and thereby to have the
ability to produce high local concentrations of
nonphosphorylated glucose within the vacuolar system. This glucose
production, in turn, could act as a transducer (e.g., metabolic,
osmotic, or cybernetic) in the multifactorial process of insulin
release. Glycogen is a normal constituent of islet tissue (11, 21), and
it is known to display a surprisingly constant concentration at a wide
range of blood glucose levels (21). Hence an important part of islet
glycogen is probably not integrated in the metabolic pool of glucose
phosphorylation processes in the cytoplasm but rather is restricted to
a compartmentalized vacuolar pool of signal glycogen available to the
acid glucan 1,4--glucosidase. It is worth noting that the
phosphorolytic breakdown of glycogen in vitro from mouse islets is
reportedly very slow (21).
In a recent report (28) we showed that the defective glucose-induced
insulin release from isolated mouse islets in a
Ca2+-deficient medium was
accompanied by markedly reduced activities of islet lysosomal
-glucosidehydrolases. In contrast, the activity of another
lysosomal glycosidase,
N-acetyl-
-D-glucosaminidase, was completely unaffected by Ca2+
deficiency. Likewise, islet activities of the lysosomal enzyme acid
phosphatase and the neutral -glucosidase (endoplasmic reticulum) were not influenced in a
Ca2+-deficient medium. A similar
pattern of a greatly suppressed glucose-induced insulin release in
parallel with a reduced acid -glucosidehydrolase activity was
accomplished by different selective -glucosidehydrolase inhibitors
such as miglitol, emiglitate, and acarbose (19, 20 28-32).
Hence, because the activity of the lysosomal acid
-glucosidehydrolases (but not the neutral -glucosidase) seemed to
be one of several important intracellular key factors in
glucose-induced insulin release, we found it mandatory to study the
Ca2+ dependency of these enzyme
activities in more detail. In the present investigation we tested
1) high concentrations of
extracellular Ca2+,
2) the
Ca2+ channel blocker nifedipine
and the intracellular Ca2+
mobilizer carbachol, as well as 3)
the selective -glucosidehydrolase inhibitor emiglitate (26) to
further elucidate the role of Ca2+
in regulating islet acid -glucosidehydrolase activities and insulin
release.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Animals
Adult female mice of the NMRI strain (B & K Universal, Sollentuna, Sweden), 3-4 mo old and weighing 25-30 g, fed ad libitum, and with free access to water, were used throughout the study. The experiments were approved by the Ethical Committee for Animal Research at the University of Lund.Drugs and Chemicals
Collagenase (CLS 4) was obtained from Worthington Biochemicals (Freehold, NJ). Nifedipine, ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic
acid (EGTA), and carbachol as well as methylumbelliferyl-coupled
substrates were purchased from Sigma Chemical (St. Louis, MO).
Emiglitate,
N-[-(4-ethoxycarbonylphenoxy) ethyl]-1-deoxynojirimycin (Bay 0 1248), was kindly supplied by Bayer (Leverkusen, Germany). Bovine serum albumin was from ICN Biomedicals (High Wycombe, UK).
45CaCl2
was from Radiochemical Centre (Amersham). All other chemicals were from
British Drug Houses (Poole, UK) or Merck (Darmstadt, Germany). The
radioimmunoassay kits for insulin determination were obtained from Novo
Nordisk (Bagsvaerd, Denmark).
Experimental Procedure
Isolation of pancreatic islets from freely fed mice was accomplished by retrograde injection of a collagenase solution via the bile-pancreatic duct (5). The animals were killed from 8 to 9 AM by elongation of the neck and were immediately injected with collagenase.Batch incubation of isolated islets. The freshly isolated islets were preincubated for 30 min at 37°C in Krebs-Ringer bicarbonate (KRB) buffer, pH 7.4, supplemented with 10 mmol/l N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 0.1% bovine serum albumin, and 1 mmol/l glucose. Each incubation vial contained 30 islets in 1.5 ml buffer solution and was gassed with 95% O2-5% CO2 to obtain constant pH and oxygenation. After preincubation, the buffer was changed to a medium containing 1 or 16.7 mmol/l glucose ± test substances, and the islets were incubated for 120 min. Emiglitate, when used, was included also during the preincubation period (20). All incubations were performed at 37°C in an incubation box (30 cycles/min). It should be noted that, in the experiments with high concentrations of Ca2+, phosphate and sulfate in the KRB-HEPES buffer were replaced with equimolar amounts of chloride (9). The change in osmotic pressure with high Ca2+ was compensated for by reduction of NaCl, as described by Hellman (9). Immediately after incubation, an aliquot of the medium was removed for assay of insulin. Insulin was determined radioimmunologically (7).
Lysosomal enzyme activities.
If not otherwise stated, the islets were then thoroughly washed in
glucose-free KRB buffer and collected and stored in 200 µl
acetate-EDTA buffer (1.1 mmol/l EDTA and 5 mmol/l acetate, pH 5.0) at
20°C (14). Ancillary experiments showed that the collected
islets could be kept frozen for several months without loss of enzyme
activity. After sonication, islet homogenates were analyzed for
lysosomal enzyme activities. In experiments in which we studied the
direct influence of different Ca2+
concentrations added to islet homogenates on the lysosomal enzyme activities, the islets were washed in a glucose- and
Ca2+-free KRB buffer and collected
and stored in 5 mmol/l acetate in the absence of EDTA. The procedures
for determination of acid phosphatase (pH 4.5), acid -glucosidase
(pH 4.0 and 5.0),
N-acetyl--D-glucosaminidase (pH 5.0), and neutral -glucosidase (pH 6.5) with
methylumbelliferyl-coupled substrates have previously been described
(16). Islet glucan 1,4--glucosidase activity with glycogen as
substrate was determined at pH 4.0, as described in detail elsewhere
(14, 19). The acid -glucosidase activity was assayed at both pH 4.0 and pH 5.0, because previous studies (23) have shown that inhibition of
-glucosidase by the lysosomotropic drug suramin was dependent on the
prevailing pH value. Furthermore, it should be recalled that lysosomal
enzyme activities are subjected to circadian and seasonal variations
(see Ref. 6). Therefore, all experiments were always performed with
both control groups and experimental groups at each occasion.
Protein was analyzed according to the method of Lowry et al.
(13).
Islet perifusion experiments. In the perifusion experiments, islets (150-200) were first incubated for 90 min in 800 µl of KRB medium containing 20 mmol/l glucose and 50 µl 45CaCl2 (50-100 µCi), which was added from a stock solution with a specific activity of 10-40 mCi/mg Ca2+. The islets were then washed three times with nonradioactive medium, divided into two or three groups with 75-100 islets per group, and transferred to perifusion columns. The islets were thereby sandwiched between two layers of gel (Bio-gel P-4, 200-400 mesh; Bio-Rad Laboratory, Richmond, CA) and perifused at a rate of 0.1 ml/min with the KRB buffer supplemented with 1 mmol/l glucose. Test substances were introduced according to the protocols. A Ca2+-deficient medium was obtained by omitting calcium chloride and adding 0.5 mmol/l EGTA. The radioactivity lost by the islets was measured in effluent fractions collected every 2 min (50 µl of the sample were added to 5 ml of scintillation fluid) and counted in a scintillation counter (Packard Instrument, Downers Grove, IL). The fractional efflux rate was calculated for each period (radioactivity lost by islets during the time interval/radioactivity present in the islets during the same time interval), and the mean value calculated for minutes 40 and 42 was then normalized to 100%. Insulin was determined with a radioimmunoassay (7).
Statistics
Probability levels of random differences were determined by Student's unpaired t-test or analysis of variance followed by Tukey-Kramer's multiple comparisons test where applicable.| |
RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Effect of a Maximal Concentration of Extracellular Ca2+ on Basal Insulin Release and Islet Lysosomal Enzyme Activities
Taking advantage of a previous dose-response study by Hellman (9) showing that increasing extracellular Ca2+ concentrations up to 30 mmol/l in the presence of a substimulatory glucose level could increase insulin release from isolated ob/ob islets, we performed a series of experiments at 1 mmol/l glucose with either a normal Ca2+ (2.5 mmol/l) or a high maximal (9) concentration of Ca2+ (30 mmol/l) in the extracellular medium. Figure 1 shows that increasing the Ca2+ concentration from 2.5 to 30 mmol/l induced an almost threefold increase in basal insulin release. This enhanced insulin secretion was accompanied by a marked increase in islet lysosomal-glucosidehydrolase activities,
i.e., acid glucan 1,4--glucosidase (3-fold increase), acid
-glucosidase pH 4.0 (+40%), and pH 5.0 (+95%), whereas
other lysosomal enzyme activities such as acid phosphatase and
N-acetyl--D-glucosaminidase were totally unaffected. In contrast, the activity of the neutral -glucosidase, an enzyme attributed to the endoplasmic reticulum, was
modestly reduced (30%) by 30 mmol/l
Ca2+ in the incubation medium.
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Effects of Direct Addition of Different Concentrations of Ca2+ on Lysosomal Enzyme Activities in Islet Homogenates
Because a high concentration of extracellular Ca2+ greatly increased the acid-glucosidehydrolase activities in intact islets (Fig. 1), we studied
whether addition of Ca2+ to islet
homogenates could influence these enzyme activities directly. The
effects of direct addition to islet homogenates of a wide range of
Ca2+ concentrations (0.05 µmol/l-30 mmol/l) on the different enzyme activities are illustrated
in the absence (Fig. 2,
A and
B) and presence (Fig. 2,
C and
D) of calmodulin. No appreciable
influence of Ca2+ was seen within
known intracellular fluctuations of the cation. However, there was a
large decrease in acid -glucosidase activities (about 80%)
and a marked increase in acid glucan 1,4--glucosidase activity
(about +50%) at very high "unphysiological" intracellular concentrations of Ca2+ (2, 10, and
30 mmol/l).
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Influence of the Ca2+ Channel Blocker Nifedipine on Islet Lysosomal Enzyme Activities and Insulin Release at Low and High Glucose Concentrations
To further investigate, in intact islets, the effect of Ca2+ perturbations on insulin release in relation to the activities of the acid-glucosidehydrolases, we studied the influence of nifedipine on
basal and glucose-induced insulin secretion and islet lysosomal enzyme
activities. Figure
3A
shows the effect of nifedipine on insulin secretion from incubated
islets at low or high glucose. As expected, glucose-induced insulin
secretion was greatly suppressed in the presence of nifedipine.
Moreover, we found, unexpectedly, that the islet lysosomal acid
-glucosidehydrolase activities were significantly increased by
nifedipine at basal glucose (Fig. 4), i.e.,
acid glucan 1,4--glucosidase (+35%) and acid -glucosidase pH 4.0 (+45%) and pH 5.0 (+40%). However, nifedipine had no effects on these
enzymes in the presence of high glucose (16.7 mmol/l), a glucose
concentration which by itself markedly enhanced the acid
-glucosidehydrolase activities (Fig. 4).
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Effects of Direct Addition of Nifedipine on Lysosomal Enzyme Activities in Islet Homogenates
To elucidate whether nifedipine could directly activate the islet-glucosidehydrolases in islet homogenates, a dose-response study was
performed. Table 1 shows the effect of
nifedipine on the activities of the different lysosomal enzymes and the
neutral -glucosidase in islet homogenates. Nifedipine at 1 µmol/l
induced a marked increase in
N-acetyl--D-glucosaminidase
activity. Furthermore, nifedipine at 30 µmol/l induced a modest
suppression of the activities of the acid -glucosidehydrolases
(about 10 to 15%) and a pronounced decrease of
N-acetyl--D-glucosaminidase
activity (about 55%). Acid phosphatase activity was not
influenced, whereas the activity of the neutral -glucosidase was
moderately reduced at 10 and 30 µmol/l nifedipine.
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Effect of Nifedipine on 45Ca2+ Efflux from Isolated Islets Either at Low (1 mmol/l) Glucose and Normal Ca2+ or in a Ca2+-Deficient Medium in Which the Islets Were Stimulated by the Intracellular Ca2+ Mobilizer Carbachol at Low Glucose
Because high extracellular Ca2+ (Fig. 1) as well as nifedipine (Fig. 4) could increase the acid-glucosidehydrolase activities in intact islets, we searched for an
effect of nifedipine on intracellular Ca2+ that was independent of its
Ca2+ channel-blocking effects.
Therefore, to study whether nifedipine could influence the efflux of
Ca2+ from the -cell, we
performed a series of perifusion experiments with
45Ca2+-loaded
islets. The first experiment was conducted at substimulatory (1 mmol/l)
glucose and normal Ca2+ (Fig.
5). It is seen that nifedipine induced a
marked decrease of
45Ca2+
efflux at this low glucose concentration, which indeed does not open
the nifedipine-sensitive Ca2+
channels. The basal insulin release was not influenced by nifedipine (see also Fig. 3A). Because the
cholinergic muscarinic receptor agonist carbachol is known to mobilize
intracellularly stored Ca2+ (12),
we performed another experiment at 1 mmol/l glucose with carbachol as a
stimulus in a Ca2+-deficient
perifusion medium supplemented with 0.5 mmol/l EGTA, to preclude any
influx of Ca2+ into the -cells.
Figure 6 shows the effect of nifedipine on 45Ca2+
efflux and insulin release during stimulation with carbachol (50 µmol/l). In the absence of nifedipine, carbachol induced a clear
biphasic increase of
45Ca2+
efflux. Addition of nifedipine converted the initial carbachol-induced increase of
45Ca2+
into a marked but transient decrease followed by a modest increase, which, however, was strongly suppressed compared with the carbachol controls. Thus nifedipine induced a powerful inhibition of
carbachol-stimulated 45Ca2+
efflux in a Ca2+-deficient medium.
As expected, carbachol did not notably influence insulin release from
either controls or nifedipine-treated islets in the absence of
extracellular Ca2+ (Fig. 6,
bottom).
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Effect of Nifedipine on Islet Lysosomal Enzyme Activities and Insulin Release in a Ca2+-Deficient Medium in the Presence and Absence of the Intracellular Ca2+ Mobilizer Carbachol
The next experiment was performed to test whether the carbachol-mobilized Ca2+, which was retained within the-cell by the action of nifedipine (see Fig.
6), might be redistributed to affect the acid -glucosidehydrolase activity. To avoid any influence of extracellular
Ca2+, the experiment was performed
in a Ca2+-deficient medium. We
therefore incubated isolated islets for 60 min in the absence and
presence of nifedipine and carbachol after a preincubation period of 40 min, i.e., largely mimicking the perifusion experiments. Figure
7 shows that nifedipine induced a large
increase (almost 2-fold) in islet lysosomal acid -glucosidehydrolase activities at basal glucose (1 mmol/l) and
Ca2+ deficiency, i.e., a much
higher increase than in the presence of a normal concentration of
extracellular Ca2+ (see Fig. 4).
The activities of other lysosomal enzymes and the neutral
-glucosidase were not influenced by nifedipine (Fig. 7). Carbachol
had no notable influence on the different enzyme activities in the
basal state and did not induce a greater increase in enzyme activities
together with nifedipine than that brought about by nifedipine itself,
except for inducing a modest but significant increase in the acid
glucan 1,4--glucosidase activity (+22%) in the presence of the
Ca2+ channel blocker (Fig. 7).
Hence, nifedipine stimulated acid glucan 1,4--glucosidase activity
in both the absence and the presence of carbachol. As expected, no
effect of either carbachol or nifedipine on insulin release in the
Ca2+-deficient medium was observed
(Fig. 3B).
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Effect of a Maximal Concentration of Extracellular
Ca2+ on
Glucose-Stimulated Insulin Release and Islet Lysosomal Enzyme
Activities in the Absence and Presence of the Selective
-Glucosidehydrolase Inhibitor Emiglitate
-glucosidehydrolases and insulin release in intact islets was
intimately coupled in the secretory process, we performed a series of
experiments with the selective -glucosidehydrolase inhibitor
emiglitate (20, 26, 29, 31) in the presence of high glucose and using
either a normal (2.5 mmol/l) or a maximal concentration of
Ca2+ (10 mmol/l). At this high
glucose concentration 10 mmol/l
Ca2+ is maximal, because higher
Ca2+ concentrations are even
inhibitory to insulin release (8). Figures
3C and 8
show that raising the extracellular
Ca2+ from 2.5 to 10 mmol/l at 16.7 mmol/l glucose (open bars) did increase both insulin release (+55%;
Fig. 3C) and acid glucan 1,4--glucosidase activity (+50%) as well as acid -glucosidase pH
4.0 (+55%) and pH 5.0 (+40%; Fig. 8). The activities of acid phosphatase and
N-acetyl--D-glucosaminidase,
however, were significantly suppressed by high
Ca2+ (25%; Fig. 8).
Moreover, addition of emiglitate almost totally suppressed both
glucose-induced and Ca2+-induced
increase of acid -glucosidehydrolase activities (Fig. 8) as well as
the release of insulin (Fig. 3C).
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The present results lend further support to our previous hypothesis
(14-20, 28-32) that one of the multiple signals involved in
glucose-induced insulin release is transduced through the activation of
a glycogen-hydrolyzing acid, glucan 1,4--glucosidase.
Effects of Ca2+ Deficiency and High Extracellular Ca2+
In a recent study we reported (28) that the activities of the acid-glucosidehydrolases were highly depressed in islets incubated in a
Ca2+-deficient medium. In
contrast, classical lysosomal enzyme activities, such as acid
phosphatase and
N-acetyl--D-glucosaminidase,
as well as the neutral -glucosidase (endoplasmic reticulum), were unaffected by the absence of extracellular
Ca2+. In addition, we observed
(28) that the presence of a high concentration of glucose in the
incubate induced a significant increase of islet acid
-glucosidehydrolase activities also in a
Ca2+-deficient medium. The
measured increase, however, never achieved the level of enzyme activity
recorded in the presence of a normal physiological
Ca2+ concentration (28). These
observations suggested to us that both glucose and
Ca2+ were needed for the full
expression of islet acid -glucosidehydrolase activity. We show here
that a high concentration of Ca2+
itself, in the presence of a substimulatory concentration of glucose (1 mmol/l), greatly enhanced the islet acid -glucosidehydrolase activities in isolated islets in parallel with an increased insulin release. These effects are most likely a result of increased
intracellular Ca2+ concentration
([Ca2+]i),
because recent data suggest that high extracellular
Ca2+ can induce a rise in
[Ca2+]i,
which primarily is accounted for by
Ca2+ influx through
dihydropyridine- and voltage-insensitive, nonselective cation channels
(33). Other lysosomal enzyme activities, such as acid phosphatase and
N-acetyl--D-glucosaminidase,
were unaffected by the high extracellular
Ca2+ level. Moreover, a maximal
(8) extracellular Ca2+
concentration at high glucose did further increase both insulin release
and the acid -glucosidehydrolase activities, supporting the idea
that Ca2+ is a key regulatory
factor for islet acid -glucosidehydrolase activities.
Effects of Blockade of Ca2+ Influx by Nifedipine and of Intracellular Ca2+ Mobilization by Carbachol
Because high extracellular Ca2+ enhanced the acid-glucosidehydrolase activity at low substimulatory
glucose (Fig. 1, present study) and because high glucose could
partially enhance the enzyme activity in the absence of extracellular
Ca2+ (28), the question arose
whether activation of the enzyme(s) was dependent on both the influx of
extracellular Ca2+ and the
redistribution/sequestration of intracellular
Ca2+. It is well established that
glucose-induced insulin release is accompanied by closure of
ATP-sensitive K+ channels,
followed by membrane depolarization (in normal mouse islets at glucose
concentrations >7 mmol/l) (4), opening of the voltage-dependent
Ca2+ channels, and influx of
Ca2+. The subsequent rise in
intracellular Ca2+ elicits
multiple signals that induce the recruitment and extrusion of secretory
granules (2, 25, 35). The present data show that nifedipine, a
well-known blocker of voltage-dependent
Ca2+ channels, unexpectedly did
induce a marked increase in acid -glucosidehydrolase activity in the
presence of a very low nondepolarizing concentration of glucose (1 mmol/l). Hence, nifedipine might have changed the intracellular
distribution of Ca2+
and/or inhibited Ca2+
outflow, because its classical
Ca2+ channel-inhibiting effects
could not be operating at 1 mmol/l glucose. Our
45Ca2+
efflux experiments showed that such an assumption was justified. Nifedipine induced a marked suppression of the basal efflux of 45Ca2+
in the presence of 1 mmol/l glucose and a normal extracellular Ca2+ concentration. This effect
was in all probability not solely, if at all, a consequence of an
inhibitory effect of nifedipine on the influx of
Ca2+ through nonvoltage-dependent
Ca2+ channels, because
45Ca2+
efflux in a Ca2+-deficient medium
with very low glucose (1 mmol/l) was strongly inhibited by nifedipine
also during stimulation by the intracellular Ca2+ mobilizer carbachol.
Carbachol has previously been shown to be a very efficient mobilizer of
intracellular Ca2+ in isolated
islets (12). Hence, in addition to its blocking effect on
voltage-dependent Ca2+ channels,
nifedipine, at least under our experimental conditions, also inhibited
the outflow of Ca2+ across the
plasma membrane and/or caused a redistribution of intracellular
Ca2+, leading to accumulation of
Ca2+ in acid
-glucosidehydrolase-containing organelles in the vacuolar system.
Such an assumption is in accordance with a very recent finding (1),
showing that a high concentration (30 µmol/l) of the nifedipine
analog nicardipine did enhance the cytoplasmic Ca2+ concentration in mouse
thymocytes in the absence of extracellular Ca2+. Indeed, a
Ca2+ redistribution effect of
nifedipine (or a nifedipine-induced messenger) rather than, or in
addition to, an inhibition of
45Ca2+
efflux across the plasma membrane, is suggested by the observation that
the initial acute increase in
45Ca2+
efflux in the biphasic response to carbachol stimulation was converted
into a marked initial decrease (negative peak; see Fig. 6) by
nifedipine followed by a modest and highly suppressed second phase.
Such a pattern is in fact much like the
45Ca2+
efflux curve obtained by glucose stimulation (3) in the presence of
extracellular Ca2+ (and in the
absence of nifedipine).
The putative Ca2+ redistribution
leading to activation of the acid -glucosidehydrolases seemed in no
way obligatory to the inflow of extracellular
Ca2+ through the voltage-dependent
Ca2+ channels, because enzyme
activity could be induced by nifedipine in a
Ca2+-deficient medium. From these
experiments it is also obvious that the nifedipine-stimulated
Ca2+ redistribution and the
subsequent increase in acid glucan 1,4--glucosidase activity are not
sufficient by themselves to induce an insulin secretory response,
because nifedipine at the same time blocks the voltage-dependent
Ca2+ channels. Furthermore, it
seems very unlikely that Ca2+
itself is directly modulating the enzyme(s), because physiological [Ca2+]i
(10, 25, 27, 33) in both the absence and presence of calmodulin did not
display any notable effect on the enzyme activities after addition to
islet homogenates. It should be noted, however, that very high
concentrations of Ca2+ (2 mmol/l-30 mmol/l) did activate considerably the acid glucan 1,4--glucosidase but inhibited the acid -glucosidases (Fig. 2).
Such high Ca2+ concentrations are
not likely to occur intracellularly, although theoretically they cannot
be completely ruled out in certain
Ca2+-rich subcellular compartments
and organelles. Furthermore, this highly differential action of 2 and
10 mmol/l Ca2+ on the acid glucan
1,4--glucosidase, compared with the acid -glucosidases
in islet homogenates, was indeed not reflected in the various
experiments with isolated intact islets, where these activities were
always increased by the supraphysiological extracellular
Ca2+ concentrations used in our
studies (see Figs. 1 and 8).
With regard to glucose-stimulated insulin release, it is conceivable
that the initial glucose-induced decrease in
45Ca2+
efflux (3), which was recently shown to be the result of the ability of
glucose to induce sequestration of cytoplasmic
Ca2+ in a slowly exchangeable
"organelle pool" (10), may be a key event in this context.
Interestingly, the glucose-induced redistribution and sequestration of
intracellular Ca2+ are manifested
earlier and at lower glucose concentrations than those required to open
the voltage-dependent Ca2+
channels (10). It is not inconceivable that one of these sequestration targets is the acid glucan 1,4--glucosidase-containing organelles. Such an assumption is in accordance with previous data showing, in a
Ca2+-deficient medium, that an
increase in glucose concentration from 1 to 4 mmol/l also increased the
acid -glucosidehydrolase activities (28). Hence, in addition to
other factors, glucose-stimulated insulin release is apparently
dependent on both a redistribution and a sequestration of intracellular
Ca2+, which in turn activate the
lysosomal-acid glucan 1,4--glucosidase system as well as the inflow
of extracellular Ca2+ through
voltage-dependent Ca2+ channels at
depolarizing glucose concentrations. This redistribution/sequestration hypothesis also conforms with recent observations (29) showing that the
acid -glucosidehydrolase activities were profoundly suppressed in
islets incubated in a
Ca2+-deficient medium in the
presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine
(IBMX), which is known to induce perturbations of intracellular
organelle-bound Ca2+ in the
-cell (2).
Effects of the Selective -Glucosidehydrolase
Inhibitor Emiglitate
-glucosidase system and glucose-induced insulin release are closely interconnected. Emiglitate almost totally
suppressed insulin release stimulated both by high glucose alone and by
high extracellular Ca2+ in the
presence of high glucose. This powerful inhibition of insulin release
was accompanied by greatly suppressed activities of the islet acid
-glucosidehydrolases. Hence, it appears that a most
important Ca2+ effect in the
stimulus-secretion coupling of glucose-stimulated insulin release is
exerted closely proximal to the action of the acid glucan
1,4--glucosidase, the inhibition of which greatly impairs the signal
transduction. This inhibition of enzyme activity and subsequent insulin
release apparently cannot be overcome by greatly increasing the
Ca2+ concentration (see Fig. 8).
Thus it is not inconceivable that this particular effect of
Ca2+ is exerted on certain
membrane components of acidic organelles and/or key factor(s)
assisting the acid -glucosidehydrolases in their in vivo catalytic
function. It should be noted that emiglitate is reportedly (26) a
selective -glucosidehydrolase inhibitor. Hence, our results suggest
a direct cause-effect relationship between islet acid glucan
1,4--glucosidase activity on the one hand and
glucose-Ca2+-induced insulin
release on the other. The present data are thus in accordance with
previous observations in our laboratory showing that nutrient-induced
insulin release is greatly suppressed by different selective
-glucosidehydrolase inhibitors, such as the pseudotetrasaccharide
acarbose or the deoxynojirimycin derivatives miglitol and emiglitate
(20, 28-32), whereas
Ca2+-independent insulin secretion
induced by IBMX is not (29). Moreover, receptor-activated insulin
release induced by carbachol is unaffected by selective
-glucosidehydrolase inhibition (30, 32). These data also
conform with the present results showing that carbachol itself had no
influence on islet acid glucan 1,4--glucosidase activity in a
Ca2+-deficient medium (see Fig.
7). In contrast, glucose has previously been shown to greatly enhance
the enzyme activity during Ca2+
deficiency (28).
In summary, in intact islets, high supraphysiological concentrations of
extracellular Ca2+ brought about a
marked enhancement of the islet acid -glucosidehydrolase activities,
accompanied by a large insulin release. The
Ca2+ channel blocker nifedipine
unexpectedly brought about an increase in acid -glucosidehydrolase
activity at low glucose. This increase was explained by showing that
nifedipine suppressed
45Ca2+
outflow from perifused islets at substimulatory glucose and normal Ca2+, as well as after
intracellular mobilization of
45Ca2+
by carbachol in a Ca2+-deficient
medium. The inhibition of
45Ca2+
efflux was probably accomplished through increased intracellular sequestration and impaired outflow of
Ca2+ across the plasma membrane.
The Ca2+-induced effects were
shown not to be exerted by a direct action of either nifedipine or
Ca2+ on the acid
-glucosidehydrolases. Instead we suggest that this signal function
of Ca2+ is exerted on a step
closely proximal to enzyme activation, e.g., on certain membrane
constituents of acidic organelles and/or key factor(s)
modulating the acid -glucosidehydrolases in their in vivo catalytic
function. This was further emphasized by the finding that selective
inhibition of the acid -glucosidehydrolases by emiglitate almost
abolished glucose-induced insulin release, an effect which could not be
overcome by increased Ca2+. Taken
together with data on islet acid -glucosidehydrolase activities
obtained from previous experiments in
Ca2+-deficient media (28), a
redistribution of Ca2+ induced by
glucose (or by pharmacological agents such as nifedipine) that is
directed to acid -glucosidehydrolase-containing organelles appears
an attractive mechanism in this context. The intimate details of
Ca2+ redistribution,
sequestration, and induction of acid glucan 1,4--glucosidase activity in nutrient-induced insulin release will await further investigations.
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ACKNOWLEDGEMENTS |
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The skillful assistance of Elsy Ling and Britt-Marie Nilsson and the secretarial help of Eva Björkbom and Björn Otterlin are gratefully acknowledged.
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FOOTNOTES |
|---|
This study was supported by the Swedish Medical Research Council (14X-4286), the Crafoord Foundation, the Swedish Diabetes Association, the Albert Påhlsson Foundation, and the Åke Wiberg Foundation.
Address for reprint requests: A. Salehi, Dept. of Pharmacology, Sölvegatan 10, S-223 62 Lund, Sweden.
Received 12 June 1997; accepted in final form 24 November 1997.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
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, 30 µmol/l) or solvent (controls, 
) was introduced at
minute 42, as shown by dotted vertical
line. Fractional efflux rate was normalized, as described in
MATERIALS AND METHODS. Values are
means ± SE for 6-7 perifusions in each group obtained from 3 independent experiments.
