Abteilung Klinische Endokrinologie, Medizinische Hochschule Hannover, 30623 Hannover, Germany
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ABSTRACT |
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Arginine vasopressin (AVP), bombesin, and
ACh increase cytosolic free Ca2+ and potentiate
glucose-induced insulin release by activating receptors linked to
phospholipase C (PLC). We examined whether tolbutamide and diazoxide,
which close or open ATP-sensitive K+ channels
(KATP channels), respectively, interact with PLC-linked Ca2+ signals in HIT-T15 and mouse -cells and with
PLC-linked insulin secretion from HIT-T15 cells. In the presence of
glucose, the PLC-linked Ca2+ signals were enhanced by
tolbutamide (3-300 µM) and inhibited by diazoxide (10-100
µM). The effects of tolbutamide and diazoxide on PLC-linked
Ca2+ signaling were mimicked by BAY K 8644 and nifedipine,
an activator and inhibitor of L-type voltage-sensitive Ca2+
channels, respectively. Neither tolbutamide nor diazoxide affected PLC-linked mobilization of internal Ca2+ or store-operated
Ca2+ influx through non-L-type Ca2+ channels.
In the absence of glucose, PLC-linked Ca2+ signals were
diminished or abolished; this effect could be partly antagonized by tolbutamide. In the presence of glucose, tolbutamide potentiated and diazoxide inhibited AVP- or bombesin-induced insulin secretion from HIT-T15 cells. Nifedipine (10 µM) blocked both the
potentiating and inhibitory actions of tolbutamide and diazoxide on
AVP-induced insulin release, respectively. In glucose-free medium,
AVP-induced insulin release was reduced but was again potentiated by
tolbutamide, whereas diazoxide caused no further inhibition. Thus
tolbutamide and diazoxide regulate both PLC-linked Ca2+
signaling and insulin secretion from pancreatic
-cells by modulating KATP channels, thereby determining voltage-sensitive
Ca2+ influx.
phospholipase C; intracellular calcium; insulin secretion; adenosine 5'-triphosphate-sensitive potassium channels
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INTRODUCTION |
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THE CONTROL OF insulin secretion is a multifactorial
and highly interconnected process involving nutritional and
nonnutritional factors. Glucose stimulates insulin secretion by an
increase in the cytosolic ratio of ATP to ADP, which inhibits
ATP-sensitive K+ channels (KATP channels),
thereby causing membrane depolarization, activation of
voltage-sensitive Ca2+ influx, and a rise in the cytosolic
free Ca2+ concentration
([Ca2+]i) that triggers insulin
secretion (19, 22, 38). In addition, glucose further augments insulin
secretion by a KATP channel-independent pathway, i.e., by
enhancing the stimulatory effect of Ca2+ on the secretory
process (7, 29, 38). Neurotransmitters and hormones that activate the
Ca2+-phosphoinositide (PI) pathway potentiate
glucose-induced insulin release and thus may be of major relevance to
the regulation of insulin secretion (27, 38, 40). ACh, arginine
vasopressin (AVP), and bombesin, which activate the Ca2+-PI
signaling pathway, cause a rise in
[Ca2+]i and stimulate insulin secretion
from both normal and transformed -cells (6, 14, 17, 24, 25, 27, 30,
32, 34, 40). The generation of Ca2+ signals by
phospholipase C (PLC)-linked hormones requires inositol 1,4,5-trisphosphate (IP3)-linked mobilization of
Ca2+ from intracellular stores and voltage-sensitive and
-insensitive Ca2+ influx from the outside (17, 30, 32). The
actions of PLC-linked agonists on [Ca2+]i
and on insulin secretion are glucose dependent, further demonstrating the interactive regulation of insulin secretion (10, 18, 27, 40).
Sulfonylurea drugs, which are widely used drugs in the treatment of
non-insulin-dependent diabetes mellitus, and diazoxide, which has been
used for many years to control hypoglycemia caused by inappropriate
insulin secretion, modulate insulin release by regulating
KATP channel activity (23). Sulfonylureas, which much like
a rise in extracellular glucose, inhibit KATP channels and
cause membrane depolarization, activation of voltage-sensitive Ca2+ influx, and a rise in
[Ca2+]i that initiates insulin release
(3, 23, 33). Diazoxide, by contrast, activates KATP
channels, thereby causing membrane hyperpolarization, inhibition of
Ca2+ influx through voltage-sensitive Ca2+
channels (VSCC), and inhibition of insulin secretion (3, 23, 33). Thus
control of insulin release by sulfonylureas or diazoxide is thought to
be mainly caused by its own regulatory actions on KATP
channels and by augmenting or opposing the effects of glucose on
KATP channel activity. However, given the pivotal role of
KATP channels in determining the membrane potential and
thereby controling voltage-sensitive Ca2+ influx in
-cells, sulfonylurea- or diazoxide-induced modulation of
KATP channel activity may also interact with the
intracellular Ca2+ signal evoked by PLC-linked hormones,
thereby regulating insulin release in response to these agonists. This
may be relevant to our understanding of the pharmacological
actions of sulfonylureas and diazoxide, and the role of
KATP channels in PLC-linked control of
-cell function,
and may elucidate some of the mechanisms underlying the glucose
dependency of PLC-linked Ca2+ signaling and insulin
secretion. In the present study, we therefore investigated the effects
of tolbutamide and diazoxide on the cytosolic Ca2+ signal
and insulin secretion evoked by the PLC-linked agonists AVP and
bombesin and the ACh analog carbachol.
[Ca2+]i was measured in single fura
2-loaded HIT-T15 and normal mouse
-cells. Insulin release was
determined from cell populations of HIT-T15 cells that respond to
various secretagogues, including glucose (12, 28).
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MATERIALS AND METHODS |
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HIT-T15 cell culture. The HIT-T15 cells were kindly provided by Dr. W. Knepel (Göttingen, Germany). The cells were grown in RPMI 1640 medium containing 10 mM glucose supplemented with 10% FCS (vol/vol), 100 units of penicillin/ml, and 100 µg streptomycin/ml at 37°C in 5% CO2 and 95% air (vol/vol). All experiments were performed with cells from passage 65 to 86.
Preparation of islet -cells.
NMRI mice were housed in a temperature-controlled room with a 12:12-h
light-dark cycle and had ad libitum access to standard chow and water.
They were treated in accordance with all guidelines and regulations of
our institutional animal care and use committee. The islets of
Langerhans were isolated from female NMRI mice aged 8-12 wk by
collagenase digestion. To obtain dispersed cells, islets were incubated
for 10 min in Ca2+-free medium [135 mM NaCl, 5.6 mM KCl,
1.2 mM MgCl2, 3 mM glucose, 10 mM NaHEPES, 100 units of
penicillin/ml, 100 µg streptomycin/ml, and 1% BSA (wt/vol) gassed
with 100% O2 (vol/vol), pH 7.4] with gentle pipetting
through a siliconized glass pipette until the islets disappeared. Islet
cells were washed, resuspended in RPMI 1640 medium containing 5.5 mM
glucose supplemented with 10% FCS (vol/vol), 100 units of
penicillin/ml, and 100 µg streptomycin/ml, allowed to attach to glass
coverslips, and maintained in a short culture for up to 2 days at
37°C in 5% CO2 and 95% air (vol/vol).
Measurement of [Ca2+]i.
HIT-T15 cells or primary islet cells subcultured on coverslips were
loaded with 5 µM fura 2-AM for 30 min at 37°C in medium containing
130 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM
MgSO4, 1.5 mM CaCl2, 10 mM glucose, 20 mM
HEPES, 2% BSA (wt/vol), and 0.1% Pluronic F-127 (wt/vol) gassed with
100% O2 (vol/vol), pH 7.4. After being loaded, the
coverslips were washed, mounted in a temperature-controlled superfusion
chamber (37°C), and placed on the stage of a Zeiss Axiovert IM 135 equipped with a ×40 Achrostigmat oil immersion objective (Zeiss,
Jena, Germany). The chamber was superfused with the same buffer as that
used for measuring insulin release at a flow rate of 0.75 ml/min.
Ca2+ measurements were done on cells of average size and
healthy appearance (round in shape, no membrane blebs). Fura 2 fluorescence from a single cell was recorded with a dual excitation
spectrofluorometer system (Deltascan 4000; Photon Technology
Instruments, Wedel, Germany).
[Ca2+]i were calculated according to the
formula [Ca2+]i = Kd × B × (R Rmin)/(Rmax
R), where R is the
ratio of fluorescence, and the dissociation constant
(Kd) = 225 nM (9). The ratio at saturating
Ca2+ concentration (Rmax), the ratio of
fluorescence at zero Ca2+ (Rmin), and the
ratio of the fluorescence intensity at 380 nm at zero and saturated
Ca2+ concentrations (B) are constants that were determined
in the superfusion chamber from solutions containing fura 2-free
acid (1 µM) and various concentrations of free Ca2+
(data not shown). All records have been corrected for autofluorescence of unloaded cells at each wavelength before the ratio was used.
Insulin secretion. HIT-T15 cells were subcultured in six-well plates at a density of 2 × 106 cells/well. After 2 days, the culture medium was removed, and the cells were washed with medium containing 130 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 1.5 mM CaCl2, 10 mM glucose, 20 mM HEPES, and 0.1% BSA (wt/vol) gassed with 100% O2 (vol/vol), pH 7.4. The cells were then preincubated for 1 h with or without glucose (10 mM) and were washed again, and fresh medium (2 ml) was added with the respective test agents. After 15 min, the supernatant was removed, and the insulin concentration was measured by RIA using a commercial kit (Kabi Diagnostics Pharmacia, Uppsala, Sweden). During the experiments, the cells were kept at 37°C.
Materials. Fura 2-AM and Pluronic F-127 were purchased from Molecular Probes (Eugene, OR), RPMI 1640, penicillin, and streptomycin were from Life Technologies (Berlin, Germany), collagenase was from Boehringer (Mannheim, Germany), thapsigargin was from Calbiochem (Bad Soden, Germany), and AVP and the other substances were from Sigma Chemical (Munich, Germany). Nifedipine and BAY K 8544 were provided by Bayer (Leverkusen, Germany). Stock solutions were prepared in water or as follows: AVP (100 µM) in 0.01 M HCl, tolbutamide (30 mM) in 150 mM NaOH, diazoxide (10 mM) and thapsigargin (5 mM) in DMSO, nifedipine and BAY K 8644 (5 mM) in ethanol.
Statistics. Unless representative tracings are shown, values are means ± SE. Statistical analysis was performed using Student's t-test for paired or unpaired data when two samples were compared. Multiple comparisons were assessed by ANOVA followed by the Student-Newman-Keuls test. P < 0.05 was considered as significantly different.
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RESULTS |
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Effects of tolbutamide and diazoxide on PLC-linked
Ca2+ signals in HIT-T15 cells.
In HIT-T15 cells [Ca2+]i averaged 138 ± 2 nM (n = 61) in the presence of glucose (10 mM). The
sulfonylurea drug tolbutamide (30 µM) increased
[Ca2+]i by 40 ± 6 nM in 26 of 36 cells.
In the remaining 10 cells, tolbutamide (30 µM) caused no changes in
[Ca2+]i. The tolbutamide-induced rise in
[Ca2+]i could be blocked by verapamil (50 µM; data not shown). AVP (1 nM) and bombesin (200 pM) caused
repetitive Ca2+ transients, as reported previously (Figs. 1
and 2; see Refs. 30-32).
In the absence of AVP or bombesin, no Ca2+ transients were
observed. Tolbutamide (3-300 µM) enhanced the frequency of the
AVP- or bombesin-induced Ca2+ transients in 13 of 17 cells
tested (Fig. 1, A and B). Tolbutamide (3-300
µM) increased the frequency of the AVP (1 nM)-induced
Ca2+ transients from 0.74 ± 0.12 to 1.41 ± 0.13 min1 (n = 7 cells, P < 0.001) and
of the bombesin (200 pM)-induced Ca2+ transients from 0.51 ± 0.06 to 1.41 ± 0.16 min
1 (n = 6 cells,
P < 0.001). As depicted in Fig. 1A, the
acceleration of the PLC-linked Ca2+ transients by
tolbutamide was concentration dependent at the level of an individual
cell. In 4 of 17 cells, tolbutamide (30 or 300 µM) switched the AVP-
or bombesin-induced Ca2+ signal to a plateau-like rise in
[Ca2+]i with cessation of the
Ca2+ transients (Fig. 1B). Tolbutamide (300 µM)
affected the AVP- or bombesin-induced Ca2+ transients in
eight of eight cells, although not all of the cells responded to lower
concentrations of tolbutamide (3 or 30 µM). The actions of
tolbutamide (3-300 µM) on the AVP- or bombesin-induced Ca2+ transients were reversible in all cells tested (Fig.
1, A and B). Diazoxide (30 or 100 µM) decreased
[Ca2+]i by 16 ± 5 nM (n = 5
of 12 cells; data not shown) in the presence of glucose (10 mM). In the remaining seven cells, diazoxide (30 or 100 µM)
caused no changes in [Ca2+]i. In 11 of 14 cells, diazoxide (10-100 µM) reduced the frequency and sometimes
the amplitude of the AVP- or bombesin-induced Ca2+
transients, whereas in 3 of 14 cells, the Ca2+ transients
ceased in the presence of diazoxide (Fig. 1, C and D). Diazoxide (100 µM, which was the highest concentration
used) either reduced the frequency or eliminated the AVP- or
bombesin-induced Ca2+ transients in all cells tested (9 cells), but 3 cells did not respond to lower concentrations of
diazoxide (10 or 30 µM). The actions of diazoxide (10-100 µM)
on the AVP- or bombesin-induced Ca2+ transients were fully
reversible in all cells tested and could be reversed by the addition of
increasing concentrations of tolbutamide (n = 13 cells, Fig.
1, C and D). When glucose was removed from the
superfusion medium, the amplitude and the frequency of the AVP-induced
Ca2+ transients dropped, and the Ca2+
transients finally ceased after ~5-60 min (Fig. 1, E and
F; n = 8 cells). In some cells (4 of 8 cells),
however, this effect of glucose deprivation was preceded by a transient
increase in the frequency of the AVP-induced Ca2+
transients and a rise in the intertransient
[Ca2+]i (Fig. 1E). Reexposure
of cells to glucose (10 mM) led to the generation of AVP-induced
Ca2+ transients (Fig. 1, E and F). The
addition of tolbutamide (3-300 µM) partly reversed the
inhibitory effect of glucose deprivation on the AVP-induced
Ca2+ transients in five of eight cells (Fig. 1E)
while in the remaining three cells tolbutamide up to a concentration of
300 µM was without any effect (Fig. 1F). In cells that
had been pretreated for 30-60 min in glucose-free medium,
tolbutamide (30 µM) caused a plateau-like increase in
[Ca2+]i by 65 ± 13 nM (n = 6
of 10 cells).
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Mechanisms of the tolbutamide- and diazoxide-induced changes of
PLC-linked Ca2+ signals in HIT-T15 cells.
The effects of tolbutamide and diazoxide on the PLC-linked
Ca2+ transients could be mimicked by the VSCC agonist BAY K
8644 and the VSCC antagonist verapamil, respectively (Fig. 2, A
and B). BAY K 8644 (1 µM) reversibly increased the
frequency and amplitude of the PLC-linked Ca2+ transients
and led to a plateau-like rise in [Ca2+]i
in six of six cells. Verapamil (50 µM) either reduced the frequency and/or amplitude of the AVP- and bombesin-induced Ca2+
transients in 10 of 18 cells by 55 ± 16 and 18 ± 9%, respectively, or stopped them in 8 of 18 cells. In addition, verapamil (50 µM) inhibited the tolbutamide-induced enhancement of the PLC-linked Ca2+ transients in three of three cells (Fig.
2C). In Ca2+-free medium, AVP caused one or two
Ca2+ transients due to mobilization of internal
Ca2+ (data not shown and Ref. 32). As shown in Table
1, neither tolbutamide (30 µM) nor
diazoxide (100 µM) changed the amplitude of the Ca2+
transients or the amount of internally released Ca2+, as
judged by the area under the curve above basal. Thapsigargin (2 µM),
which is a major tool to study capacitative Ca2+ entry
(35), caused a biphasic rise in [Ca2+]i
with an initial peak reflecting mobilization of internal
Ca2+ and a secondary plateau phase that is caused by influx
of Ca2+ through L-type VSCC and non-L-type Ca2+
channels, most likely voltage-insensitive Ca2+ channels
(data not shown and Refs. 30 and 32). To eliminate Ca2+
influx through L-type VSCC, HIT-T15 cells were stimulated with thapsigargin (2 µM) in the presence of verapamil (50 µM). Neither tolbutamide (30 µM) nor diazoxide (100 µM) affected the
initial peak or plateau of the thapsigargin (2 µM)-induced
Ca2+ signal (Table 1).
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Effects of tolbutamide and diazoxide on PLC-linked
Ca2+ signals in mouse -cells.
In mouse
-cells, [Ca2+]i was 101 ± 5 nM (n = 32) in the presence of glucose (6 mM). Carbachol (3 µM), which stimulates muscarinic receptors coupled to the
Ca2+-PI signaling pathway, elicited a biphasic rise in
[Ca2+]i with an initial peak followed by
a sustained plateau in most cells (Fig. 3).
In some cells, however, repetitive Ca2+ transients were
observed in response to carbachol (3 µM; data not shown). Carbachol
(3 µM) increased [Ca2+]i by 327 ± 47 and 58 ± 14 nM at its peak or plateau (measured after 5 min),
respectively (n = 18). Reexposure of cells to carbachol (3 µM) after a washout period of 30 min caused a nearly identical Ca2+ response (96 ± 4% of the initial peak and
plateau; n = 9; Fig. 3A). To assess the effect of
tolbutamide on the carbachol-induced Ca2+ signal,
tolbutamide (1 µM) was added to the perfusion medium 5 min before the
second stimulation with carbachol (3 µM). Tolbutamide (1 µM)
increased [Ca2+]i by 325 ± 170 nM in
four of eight cells, whereas in the remaining four cells tolbutamide (1 µM) caused no changes in [Ca2+]i.
Pretreatment with tolbutamide (1 µM) enhanced the carbachol (3 µM)-induced Ca2+ signal when compared with the first
stimulation in the same cell (Fig. 3C). In the presence of
tolbutamide (1 µM), the carbachol (3 µM)-induced peak increase and
plateau rise in [Ca2+]i amounted to 196 ± 44 and 359 ± 155% of the control stimulation (n = 8;
P < 0.05). Diazoxide (100 µM) caused no changes in
[Ca2+]i in the presence of glucose (6 mM). Pretreatment for 5 min with diazoxide (100 µM) reduced the
carbachol (3 µM)-induced peak increase and plateau rise in
[Ca2+]i by 69 ± 13 and 68 ± 18%
(n = 4; P < 0.02; Fig. 3E),
respectively, when compared with the control stimulation in the same
cell. Preincubation of mouse
-cells for 30-60 min in
glucose-free medium reduced basal [Ca2+]i
to 86 ± 3 nM (n = 11). Under these glucose-free conditions, the carbachol (3 µM)-induced Ca2+ response was much
smaller than in the presence of glucose (6 mM) and amounted to 33 ± 10 and 10 ± 4 nM at its peak or plateau, respectively
(n = 11; Fig. 3B). Pretreatment with tolbutamide (10 µM) for 5 min, which by itself caused no changes in
[Ca2+]i, significantly enhanced the
Ca2+ signal evoked by a second stimulation with carbachol
(3 µM) in the same cells (Fig. 3B). The carbachol (3 µM)-induced peak Ca2+ response was 123 ± 54 nM, and the
plateau rise was 46 ± 31 nM (n = 4; P < 0.05).
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Mechanisms of the tolbutamide- and diazoxide-induced changes of
PLC-linked Ca2+ signals in mouse
-cells.
The effects of tolbutamide and diazoxide on the carbachol (3 µM)-induced increase in [Ca2+]i in 6 mM
glucose could be mimicked by BAY K 8644 and the VSCC antagonist
nifedipine (Fig. 3, D and F). BAY K 8644 (1 µM),
which caused a small rise in [Ca2+]i by 6 ± 2 nM (n = 7) in 6 mM glucose, increased the carbachol (3 µM)-induced peak increase and plateau rise in
[Ca2+]i to 421 ± 127 and 515 ± 154%
of the control stimulation (n = 7; P < 0.05; Fig.
3D). Nifedipine (10 µM), which like diazoxide (100 µM)
had no effect on [Ca2+]i in the presence
of glucose (6 mM), inhibited the carbachol (3 µM)-induced peak
increase and plateau rise in [Ca2+]i by
61 ± 12 and 42 ± 22% (n = 8; P < 0.05; Fig.
3F), respectively. As shown in Table
2, neither tolbutamide (30 µM) nor
diazoxide (100 µM) affected the carbachol (10 µM)-induced
Ca2+ signal in Ca2+-free medium or the
thapsigargin (2 µM)-induced Ca2+ influx through
non-L-type Ca2+ channels.
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Effects of tolbutamide and diazoxide on PLC-linked insulin secretion
from HIT-T15 cells.
In the presence of glucose (10 mM), insulin secretion was 46 ± 5 µU/ml (n = 24) during 15 min static incubation from
populations of HIT-T15 cells and was enhanced by
tolbutamide (30 µM), AVP (1 nM), and bombesin (100 pM; Fig.
4A). When tolbutamide (30 µM) and AVP (1 nM) or bombesin (100 pM) were added together, the insulin secretory response to the combined agonists was greater than additive (Fig. 4A). Diazoxide (30 µM) inhibited insulin release in
the presence of glucose (10 mM) by 20 ± 9%
(n = 12; data not significant) and diminished the AVP (1 nM)-
and bombesin (100 pM)-induced insulin secretion (Fig. 4A).
Nifedipine (10 µM) reduced insulin secretion in the presence of
glucose (10 mM) by 32 ± 4% (n = 16; P < 0.05) and inhibited AVP-induced insulin secretion by 39 ± 2%
(n = 24; P < 0.001). Neither tolbutamide (30 µM) nor diazoxide (30 µM) affected basal insulin secretion or
AVP-stimulated insulin secretion in the presence of nifedipine (10 µM), as depicted in Fig. 4B. When HIT-T15 cells had been
preincubated for 60 min in glucose-free medium and were kept at zero
glucose during the static incubation period, insulin release amounted
to 35 ± 5 µU/ml (n = 12), which was somewhat but not
significantly lower than in the presence of glucose (10 mM).
Stimulation with glucose (10 mM) for 15 min in HIT-T15 cells that had
previously been kept in zero glucose for 30-60 min resulted in a
significant increase in insulin secretion from 31 ± 2 µU/ml
(n = 29) to 51 ± 4 µU/ml (n = 23,
P < 0.009), demonstrating that the HIT-T15 cells used are
responsive to glucose. Under glucose-free conditions, the stimulation
of insulin release by tolbutamide (30 µM) and AVP (1 nM) was
significantly reduced (Fig. 4C). However, insulin secretion
in response to the combination of tolbutamide (30 µM) and AVP (1 nM)
was again greater than additive, although the overall secretory
response was lower than in experiments carried out in the presence of
10 mM glucose (Fig. 4A). In the absence of glucose, diazoxide
(30 µM) neither affected basal nor AVP (1 nM)-induced insulin
secretion from HIT-T15 cells (Fig. 4C).
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DISCUSSION |
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In the present study, we demonstrate that tolbutamide and diazoxide,
which close and open KATP channels, respectively,
critically regulate PLC-linked Ca2+ signaling in clonal
HIT-T15 and primary mouse -cells. In HIT-T15 cells, tolbutamide
caused a concentration-dependent increase in the frequency of the AVP-
or bombesin-induced Ca2+ transients in the presence of high
extracellular glucose (10 mM). At intermediate or high concentrations
of tolbutamide (30 and 300 µM), the oscillatory Ca2+
signal switched to a plateau-like rise in
[Ca2+]i. Conversely, diazoxide, which
activates KATP channels, reduced the frequency and
sometimes the amplitude of the Ca2+ transients and stopped
them completely in some cells, as described previously (16).
Qualitatively similar results were obtained in mouse
-cells where in
the presence of glucose (6 mM) tolbutamide and diazoxide potentiated or
inhibited the carbachol-induced biphasic increase in
[Ca2+]i, respectively. Because BAY K
8644, which increases the open probability of L-type VSCC, and the
L-type VSCC antagonists verapamil and nifedipine mimicked the actions
of tolbutamide and diazoxide, respectively, their effects on PLC-linked
Ca2+ signals could be explained by modulation of
voltage-sensitive Ca2+ influx.
In the presence of elevated glucose, PLC-linked agonists by themselves
cause membrane depolarization and activation of Ca2+ influx
through VSCC, which is necessary for the sustained generation of
PLC-linked Ca2+ signals in -cells, as demonstrated by
the actions of the VSCC blockers in this study and as reported
previously (16, 30-32). However, the mechanisms, underlying the
depolarizing action of PLC-linked agonists in
-cells are
incompletely understood. In RINm5F cells, AVP causes membrane
depolarization directly (21), and activation of protein kinase C has
been shown to cause membrane depolarization by closure of
KATP channels (37). In HIT-T15 cells and in normal mouse
-cells, no such direct effects of PLC-linked agonists on
KATP channels have been reported (6, 13). It rather appears
that emptying of intracellular Ca2+ stores activates a
depolarizing current, which enhances Ca2+ influx through
VSCC in
-cells (39). In addition, PLC-linked agonists may inhibit
Ca2+-activated K+ channels, thereby augmenting
glucose-induced electrical activity and voltage-sensitive
Ca2+ influx (36). Tolbutamide and diazoxide, by modulating
KATP channel activity, may therefore indirectly enhance or
oppose the depolarizing mechanisms activated by PLC-linked agonists,
thereby tuning voltage-sensitive Ca2+ influx activated by
PLC-linked agonists.
The mechanisms by which Ca2+ influx through VSCC could
modulate PLC-linked Ca2+ signals are yet unclear but may
involve modulation of IP3-production and/or the
IP3-linked Ca2+ release process (1, 2). This
can even occur without changing the average
[Ca2+]i as observed here where the
changes in PLC-linked Ca2+ signals caused by the addition
of tolbutamide or diazoxide were not usually accompanied by concomitant
changes in baseline Ca2+. Rather, such limited
Ca2+ influx may cause local changes of Ca2+ at
the microdomains adjacent to the Ca2+ regulatory sites of
the IP3 receptor, thereby determining the threshold for
IP3-mediated Ca2+ release (8). Such a mechanism
could well explain the influence of voltage-sensitive Ca2+
influx on PLC-linked Ca2+ transient frequency and amplitude
in HIT-T15 cells and on the carbachol-induced Ca2+ signal
in mouse -cells where not only the plateau of the Ca2+
signal, which is dependent on Ca2+ influx from the outside,
but also the initial peak, which mainly reflects internal
Ca2+ mobilization, is affected by tolbutamide and
diazoxide. Evidence for a direct interaction of tolbutamide or
diazoxide with IP3-induced Ca2+ mobilization
could not be found. Likewise, as judged by the actions of thapsigargin
in the presence of L-type VSCC blockers, capacitative Ca2+
entry through non-L-type Ca2+ channels, which also
contributes to sustained PLC-linked Ca2+ signals in
-cells (16, 30, 32), is not altered by tolbutamide or diazoxide.
Thus it appears that tolbutamide and diazoxide regulate PLC-linked
Ca2+ signals predominantly if not exclusively by modulating
KATP channel activity, thereby determining membrane
potential and voltage-sensitive Ca2+ influx.
Physiologically, KATP channel activity is controlled by the
cytosolic ratio of ATP to ADP, which in turn is determined by the
ambient glucose concentration. In glucose-free medium, which leads to a
decrease in the cytosolic ratio of ATP to ADP, activation of
KATP channels, and membrane hyperpolarization, the
frequency and amplitude of AVP-induced Ca2+ transients
decreased and finally ceased in HIT-T15 cells, and in mouse -cells
the carbachol-induced Ca2+ signal was greatly reduced,
confirming the glucose dependency of PLC-linked Ca2+
signaling in
-cells (10, 18, 27, 40). In the absence of glucose,
tolbutamide restored AVP-induced Ca2+ transients in some
but not all HIT-T15 cells and potentiated the Ca2+ signal
elicited by carbachol in mouse
-cells. This indicates that
glucose-dependent membrane predepolarization and facilitation of
voltage-sensitive Ca2+ influx, which is required for
further membrane depolarization and activation of VSCC by PLC-linked
signals (18), may contribute to the glucose dependency of PLC-linked
Ca2+ signals in
-cells (10, 18, 27, 40). However, other
sites appear to exist whereby glucose-dependent processes regulate
PLC-linked Ca2+ signals. Diazoxide even at high
concentrations stopped the Ca2+ transients only in a small
subset of cells, and tolbutamide only partly restored the PLC-linked
Ca2+ signals in glucose-free medium. Although not tested
here, glucose has been shown to enhance production of IP3
in response to receptor activation (40) and to be a prerequisite for
the reuptake of Ca2+ into the IP3-sensitive
Ca2+ pool (11), thus providing additional mechanisms for
the glucose dependency of the effects of PLC-linked agonists on
[Ca2+]i in
-cells.
In HIT-T15 cells, tolbutamide stimulated insulin secretion and
potentiated insulin release in response to AVP and bombesin, whereas
diazoxide reduced basal insulin release, albeit not significantly, and
inhibited PLC-linked insulin secretion in the presence of glucose (10 mM). The actions of tolbutamide and diazoxide on PLC-linked insulin
secretion largely paralleled their effects on PLC-linked Ca2+ signaling and were similar to the effects of VSCC
activation or inhibition reported previously (31). This suggests that
tolbutamide and diazoxide critically determine insulin secretion in
response to PLC-linked hormones by regulating PLC-linked
Ca2+ signaling through modulation of voltage-sensitive
Ca2+ influx. Recently, it has been proposed that
sulfonylureas and diazoxide, in addition to their actions on
KATP channels, could modulate insulin secretion by altering
the efficacy of Ca2+ on the exocytotic process (4, 5, 26).
However, such a KATP channel-independent pathway does not
appear to be involved in the actions of tolbutamide or diazoxide on
PLC-linked insulin secretion, since in the presence of nifedipine
tolbutamide failed to potentiate AVP-induced insulin secretion and
diazoxide did not further inhibit AVP-induced insulin secretion. This
is consistent with a recent report showing convincingly that both
agents influence insulin secretion by changing the concentration but
not the action of cytoplasmic Ca2+ in mouse -cells (20).
In the absence of glucose, insulin release was somewhat lower than from
cells chronically exposed to glucose (10 mM), and AVP-induced insulin
secretion was largely inhibited, confirming the glucose-dependent
action of PLC-linked agonists on insulin secretion both from
transformed and normal -cells (40). This may involve modulation of
KATP channels, since diazoxide inhibited AVP-induced
insulin secretion in the presence of glucose (10 mM) to a similar
degree and since AVP-induced insulin secretion was potentiated by
tolbutamide in the absence of glucose. However, in glucose-free medium,
the tolbutamide-induced insulin secretion was lower despite similar
effects on [Ca2+]i in the presence or
absence of glucose, and the combined effect of tolbutamide and AVP on
insulin secretion was reduced. This points to additional sites by which
glucose could enhance PLC-linked insulin secretion, such as the release
process itself (15, 38).
In summary, we demonstrate here that tolbutamide and diazoxide, by
modulating KATP channel activity and thereby controlling Ca2+ influx through VSCC, critically regulate
Ca2+ signaling and insulin secretion elicited by PLC-linked
agonists in -cells. This might contribute to the stimulatory and
inhibitory actions on insulin secretion by sulfonylureas and diazoxide
when used as therapeutic agents. Furthermore, besides their
well-accepted role in glucose-mediated Ca2+ signaling and
insulin secretion, KATP channels may be similarly important
for the regulation of insulin release in response to neurohumoral
signals activating the Ca2+-PI signaling
pathway in
-cells. Modulation of KATP channels appears
to be a major mechanism for the glucose-dependent action of PLC-linked
agonists on [Ca2+]i and insulin
secretion. This may be important for the integration of metabolic and
neurohumoral signals under conditions where the glucose concentration
is high and
-cells are exposed to PLC-linked stimuli.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Petra Wübbolt, Natalie Wittner, and Daniela Biegert for excellent technical assistance.
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FOOTNOTES |
---|
This work was supported by DFG Grant Scho 466/1-3.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: C. Schöfl, Abteilung Klinische Endokrinologie, Medizinische Hochschule, 30623 Hannover, Germany (E-mail: schefl.christof{at}mh-hannover.de).
Received 27 April 1999; accepted in final form 8 November 1999.
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