Muscarinic modulation of voltage-dependent
Ca2+ channels in
insulin-secreting HIT-T15 cells
Jeffrey A.
Love,
Neil W.
Richards,
Chung
Owyang, and
David C.
Dawson
Department of Pharmacology and Toxicology, University of Mississippi
Medical Center, Jackson, Mississippi 39216; and Departments of
Internal Medicine and Physiology, University of Michigan Medical
School, Ann Arbor, Michigan 48109
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ABSTRACT |
Potentiation of insulin secretion from
pancreatic
-cells by acetylcholine requires ongoing cyclic
electrical activity initiated by other depolarizing secretagogues.
Patch-clamp recordings in glucose-free solutions were made from the
clonal
-cell line HIT-T15 to determine whether the muscarinic
agonist bethanechol (BCh) modulated voltage-dependent
Ca2+ channels independent of
effects on membrane potential. Only high-threshold, dihydropyridine-sensitive (L-type)
Ca2+ channels with a mean
conductance of 26 pS were observed in cell-attached patches. BCh (100 µM) caused a two- to threefold increase in both fractional open time
and mean current of single Ca2+
channels. These changes resulted from a 44% decrease in the longer of
two apparent mean closed times and a 25% increase in the mean open
time. Similar BCh-stimulated increases in macroscopic
Ca2+ currents were recorded in
whole cell, perforated-patch recordings. The role of protein kinase C
(PKC) in the muscarinic activation of
Ca2+ channels was tested using a
variety of PKC activators and inhibitors. Acute application of either
the active phorbol ester phorbol 12-myristate 13-acetate (PMA) or the
membrane-permeable diacylglycerol analog 1,2-didecanoyl-rac-glycerol mimicked
the effects of BCh, whereas an inactive phorbol (4
) had no effect.
Depletion of PKC activity by chronic exposure to PMA or acute
application of the PKC inhibitor staurosporine greatly reduced or
abolished muscarinic activation of
Ca2+ channels. These results are
consistent with muscarinic activation of L-type, voltage-dependent
Ca2+ channels mediated in large
part by PKC.
pancreatic
-cells; bethanechol; diacylglycerol; protein kinase
C; L-type calcium channels
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INTRODUCTION |
PANCREATIC
-cells respond to increasing
concentrations of extracellular glucose with gradual membrane
depolarization until, at 6-8 mM glucose, a threshold is reached
for the initiation of cyclic electrical activity. This activity
consists of prolonged, voltage-dependent action potentials (plateau
potentials) with superimposed spikes, both of which are
Ca2+ dependent, interposed between
repolarized, silent phases. Both the duration of the plateau potentials
and the fraction of a given cycle spent at this potential are strongly
glucose dependent and show a close correlation with
Ca2+ influx and insulin secretion
(20). Subsequent patch-clamp studies have revealed that glucose
metabolism results in the inhibition of ATP-sensitive
K+ channels, leading to membrane
depolarization, activation of high-threshold voltage-dependent Ca2+ channels,
and Ca2+ influx essential for
stimulus-secretion coupling (2, 3, 36).
Acetylcholine (ACh) alone has little effect on insulin secretion and
-cell electrical activity yet potentiates insulin secretion in the
presence ongoing cyclic electrical activity initiated by glucose or
other depolarizing secretagogues. The secretory effects are paralleled
by both a depolarization of the plateau potentials and an increased
frequency of these Ca2+-dependent
potentials as well as the superimposed
Ca2+ spikes (6, 9, 14). Because
glucose-stimulated
-cell electrical activity, insulin secretion, and
the effects of ACh on them persist in tetrodotoxin- and
Na+-free solutions, a significant
part of the ACh effect is likely to be due to an increase in
Ca2+ channel activity (11-13,
15, 19, 20, 26, 27). Although muscarinic depolarization of
-cells
resulting from a decrease in resting
K+ conductance has been reported
(27) and could explain the increased Ca2+ influx and insulin secretion,
the possibility of a membrane potential-independent activation of
Ca2+ channels by muscarinic
agonists has not been studied in detail. Such a direct activation of
-cell Ca2+ currents has been
demonstrated for agents that increase intracellular adenosine
3',5'-cyclic monophosphate (cAMP) levels (1).
The mechanism of cholinergic modulation of
-cell electrical activity
and its relationship to insulin secretion are poorly understood.
However, it is well established that ACh, acting via G protein-coupled
muscarinic receptors, stimulates phospholipid hydrolysis, yielding
inositol trisphosphate (IP3) and
diacylglycerol (DAG) (34). The activation of protein kinase C (PKC) by
DAG results in the phosphorylation of endogenous protein substrates (8,
10) and appears to be required for the sustained,
Ca2+-dependent cholinergic
potentiation of glucose-stimulated insulin secretion (16, 22, 23). In
contrast, IP3 production causes only a transient stimulation of insulin secretion, and only at high
concentrations of cholinergic agonist (34). Protein kinase A, which is
not stimulated by ACh, potentiates glucose-stimulated electrical
activity and insulin secretion in a similar manner. These effects have
been attributed to both an increased
Ca2+ channel activity at a given
membrane potential and a sensitization of the secretory process to
Ca2+ (1, 24).
These observations led us to hypothesize that ACh, acting via PKC,
could alter the gating of voltage-dependent
Ca2+ channels activated by
membrane depolarization. To test this hypothesis, we used the clonal
-cell line HIT-T15. This continuous cell line provided a homogeneous
population of insulin-secreting cells that exhibited all the salient
electrophysiological and biochemical features of pancreatic
-cells
(15-17, 25). Cell-attached patch recordings of single
Ca2+ channel currents and
permeabilized-patch recordings of whole cell currents were used to
study the effects of a muscarinic agonist under conditions that
preserved the cytoplasmic integrity of the cells and allowed the
control of membrane potential.
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METHODS |
Cell culture.
HIT-T15 cells were obtained from the American Type Culture Collection
and grown in RPMI-1640 containing 11.1 mM glucose and supplemented with
10% fetal bovine serum, glutamine (2 mM), penicillin (100 U/ml), and
streptomycin (100 µg/ml). Cultures were maintained in a warm
(37°C), humidified atmosphere containing 95% air-5% CO2. Cells
(passages 59-75) were grown on
glass coverslips for patch-clamp recordings. At least 30 min before
patch-clamp recordings, coverslips were removed from the culture medium
and placed in a glucose-free bath solution containing (in mM) 135 NaCl,
5 KCl, 2 MgCl2, 5 CaCl2, and 5 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-NaOH (pH 7.4). This ensured that any residual effects of
glucose on Ca2+ channel activity
(3, 31) were avoided.
Patch-clamp recordings.
Single Ca2+ channel currents were
recorded from cell-attached patches in a glucose-free bath solution
containing (in mM) 115 KCl, 28 KOH, 1 MgCl2, 1 CaCl2, 10 ethylene
glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic
acid, and 10 HEPES (pH 7.4). This solution closely approximates the
cytoplasmic K+ concentration and
depolarizes the cells to a membrane potential of ~0 mV. Under these
conditions, any depolarization resulting from application of BCh was
prevented, and changes in channel activity were ascribed to a membrane
potential-independent change in channel kinetics. Gigaohm seals were
made using polystyrene-coated glass (Corning 7052) pipettes containing
a solution of (in mM) 100 BaCl2,
10 tetraethylammonium chloride (TEA · Cl), and 10 HEPES-Ba(OH)2 (pH 7.4). This
solution blocked all K+ currents
in the patches while allowing Ba2+
to serve as the carrier of inward current and minimizing the Ca2+-dependent inactivation of
channel activity reported in HIT cells (17). Membrane patches were
voltage clamped using an Axopatch-1B amplifier (Axon Instruments,
Foster City, CA), and the records were stored on computer hard disk.
Currents were filtered at 1-3 kHz using an eight-pole Bessel
filter and a digitization rate of 5-15 kHz. Capacity and leakage
currents associated with voltage steps were removed by subtracting the
composite null sweep. As has been observed in primary cultures of
-cells (31), the brief nature of the
Ca2+ channel openings combined
with their low open probability prevented us from reliably establishing
the number of channels in a patch. Because calculation of open-state
probability presumes recordings from a single channel, we instead used
fractional open time (Fo) as an
index of Ca2+ channel activation.
Single-channel currents were analyzed using pCLAMP software (Axon
Instruments) with Fo calculated
from the total open time of the channels divided by the total time of
the record and the mean current
(Iavg)
calculated from the sum of single-channel currents divided by the total
time of the record. Mean open and closed times were calculated from the
time constants of the exponential functions fitted to the frequency
distributions of open and closed times. All values were expressed as
means ± SE, and statistical comparisons were made using two-tailed,
paired Student's t-tests.
Macroscopic Ca2+ currents were
recorded using the perforated-patch variation of whole cell recording.
Amphotericin was dissolved in dimethyl sulfoxide (DMSO, 6 mg/100 µl)
and then diluted in the pipette solution to a final concentration of
240 µg/ml. The pipette solution contained (in mM) 55 CsCl, 70 Cs2SO4,
7 MgCl2, and 10 HEPES (pH 7.4),
and the bath solution contained (in mM) 136 NaCl, 10 BaCl2, or
CaCl2 (pH 7.4). After formation of
gigaohm seals, perforation began within 5 min and stabilized within
15-30 min. Only patches with stable access resistances of
8-25 M
were studied further. Whole cell currents were studied
at membrane potentials from
100 to +50 mV using a sequence of
voltage steps (10 mV; 200-500 ms) from a holding potential of
70 mV. Subtraction of linear leak currents extrapolated from
currents measured at potentials negative to the holding potential was
used to correct the observed values of macroscopic
Ca2+ currents.
Chemicals.
All pharmacological agents were ultimately dissolved in the bath
solution described above. Concentrated stocks of lipophilic compounds
were made using DMSO and diluted to yield a final concentration 0.01-0.1% DMSO. Chemical reagents included bethanechol (BCh), phorbol 12-myristate 13-acetate (PMA),
1,2-didecanoyl-rac-glycerol (DC10), 4
-phorbol, pertussis
toxin (PTX), and TEA · Cl, all purchased from Sigma
Chemical (St. Louis, MO). The dihydropyridine compounds BAY-K-8644 and
nifedipine were obtained from Research Biochemicals (Natick, MA).
 |
RESULTS |
Single Ca2+
channel currents in cell-attached patches.
Cell-attached patches exhibited single
Ca2+ channel
(Ba2+) currents in over 85% of
the cells tested. After seal formation, the patches were hyperpolarized
to pipette potentials of
60 to
100 mV, and channels were
activated by 50- to 250-ms depolarizing voltage steps to pipette
potentials up to +10 mV. Brief, low-frequency channel openings with a
mean single-channel conductance of 26 ± 1 pS
(n = 11) were observed only at pipette
potentials positive to
40 mV (Fig.
1). Kinetic analysis of the channel
openings revealed a single apparent mean open time
(
o) of 0.5 ± 0.1 ms and
two apparent mean closed times
(
c1,
c2)
of 1.3 ± 0.2 and 32.7 ± 6.5 ms, respectively
(n = 11). Marked increases in
Fo occurred only at pipette
potentials positive to
20 mV (Fig.
2), and the threshold for these channel
openings was unaffected by increasing the holding potential to values
up to
100 mV. In addition, these channels exhibited little
inactivation during voltage steps lasting several hundred milliseconds
to several seconds. Excision of the patches to form inside-out patches
resulted in a complete loss of activity within several minutes.

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Fig. 1.
Activation of single Ca2+ channel
currents in a cell-attached patch from an HIT-T15 cell. Single-channel
currents were recorded in a high (143 mM)-K+ bath solution, using
pipettes filled with a solution that contained 100 mM
BaCl2 and 10 mM
TEA · Cl. Pipette potential was stepped from a
holding potential of 90 mV to potentials indicated at
right. In all patches tested,
threshold for channel activation was positive to 40 mV, with
most channel activation occurring positive to 20 mV. Channel
openings were observed throughout duration of voltage steps, and
simultaneous opening of at least 2 channels occurred at a step
potential of 0 mV.
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Fig. 2.
HIT-T15 cells exhibited only high-threshold, voltage-dependent
Ca2+ channels. In cell-attached
patches, channel openings were recorded during a series of 10-mV
depolarizing voltage steps from a holding potential of 100 mV,
using solutions described in Fig. 1 legend. Threshold for channel
activation occurred positive to 40 mV, with most of channel
activity occurring at pipette potentials positive to 20 mV.
Fractional open time (Fo) was
fraction of total trial time (20 67-ms sweeps at each test potential)
during which at least 1 channel was open. Each point represents mean ± SE of Fo recorded from 6 patches.
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We also tested the dihydropyridine sensitivity of these high-threshold
Ca2+ channels. Bath application of
the dihydropyridine agonist BAY-K-8644 (0.1-1 µM) caused a
dramatic change in the Ca2+
channel gating pattern to one of frequent, prolonged openings (Fig.
3). This resulted in a significant increase
in Iavg from 0.04 ± 0.01 to 0.56 ± 0.19 pA
(P
0.05;
n = 5). In contrast, the
dihydropyridine antagonist nifedipine (1-5 µM) significantly reduced Iavg from
0.05 ± 0.01 to 0.01 ± 0.01 pA
(P
0.05;
n = 6), and the remaining channel
currents exhibited the same thresholds and single-channel conductances
recorded in the absence of the antagonist (Fig.
4).

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Fig. 3.
Dihydropyridine agonist BAY-K-8644 increased high-threshold
Ca2+ channel currents of HIT-T15
cells. Top: channel openings recorded
from a cell-attached patch were elicited by stepping from a holding
potential of 90 mV to a test potential of 30 mV. Brief,
infrequent channel openings occurred throughout 200-ms steps.
Bottom: 5 min after bath solution was
changed to one containing BAY-K-8644 (1 µM), channel gating was
characterized by prolonged openings that revealed at least 2 distinct
channels within the patch. Mean current
(Iavg),
calculated from 100 sweeps, increased from 0.03 to 0.78 pA/ms in
presence of agonist.
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Fig. 4.
Dihydropyridine antagonist nifedipine inhibited high-threshold
Ca2+ channel currents of HIT-T15
cells. Top: channel openings recorded
from a cell-attached patch were elicited by stepping from a holding
potential of 70 mV to a test potential of 20 mV.
Bottom: 10 min after switch to a
solution that contained nifedipine (5 µM),
Iavg, calculated
from 100 sweeps, decreased from 0.06 to 0.01 pA/ms.
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Macroscopic
Ca2+ channel
currents in permeabilized patches.
Whole cell recordings in the permeabilized-patch configuration were
used to confirm that the properties of the single
Ca2+ channel currents accurately
reflected the properties of macroscopic Ca2+ channel currents. Like the
single-channel currents, whole cell currents exhibited a threshold
positive to
40 mV, with over 80% of the maximal current
recorded at potentials positive to
20 mV (Fig. 6,
A and
B). The threshold was not changed by
increasing the holding potential from
70 to
100 mV. The
mean peak current of 105 ± 13 pA
(n = 6) occurred at +10 mV and
reversed at +50 mV. Application of nifedipine (5 µM) significantly
decreased mean peak current by 84% (from 91 ± 12 to 15 ± 4 pA)
in all three cells tested.
Effects of BCh on
Ca2+ channel
currents.
Single Ca2+ channel currents were
activated by depolarizing voltage steps (40-80 mV; 0.1-0.5
Hz) from a holding potential of
70 mV. Under control conditions,
single Ca2+ channel openings of
HIT-T15 cells occurred as low-frequency bursts described by
o,
c1, and
c2. Exposure of HIT-T15 cells
to the muscarinic agonist BCh (100 µM) caused a large increase in the frequency of channel openings (Fig. 5) in
27 of 31 patches tested. Statistically significant
(P
0.05) increases in
o,
Fo, and Iavg, as well as
significant decreases in the longer of two apparent mean closed times,
c2, accompanied the increased
activity (Table 1). These effects occurred
within 5 min of changing the bath solution and persisted throughout the
duration of exposure (tested up to 45 min after completion of bath
change). In contrast, all cells exposed to BCh after pretreatment with
atropine (10 µM; n = 6) exhibited no
significant changes in Fo (0.01 vs. 0.01) or Iavg
(0.01 vs. 0.01 pA/ms), whereas, in the same passage of cells, two of
three cells responded to BCh in the absence of atropine with
significant increases in Fo (0.02 vs. 0.06) and
Iavg (0.02 vs.
0.06 pA/ms). Pretreatment with PTX (100 ng/ml) for 12-48 h before
recording had no effect on the BCh response. In four of five cells
tested, BCh (100 µM) caused significant increases in Fo (0.03 vs. 0.07) and
Iavg (0.04 vs.
0.08 pA/ms). In recordings from cells of the same passage not treated
with PTX, BCh significantly increased
Fo (0.03 vs. 0.08) and
Iavg (0.04 vs.
0.08 pA/ms) in three of four cells tested.

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Fig. 5.
Muscarinic agonist bethanechol (BCh) increased high-threshold, single
Ca2+ channel currents independent
of a change in membrane potential.
Top: single-channel currents were
recorded from a cell-attached patch in a
high-K+ bath solution that
chemically clamped membrane potential at or close to 0 mV. Channel
openings were elicited by depolarizing voltage steps from a holding
potential of 70 mV to a test potential of 15 mV. Under
these conditions, channel openings were brief and infrequent.
Bottom: 10-min exposure to a bath
solution that contained 100 µM BCh markedly increased
Ca2+ channel activity, and
Iavg, calculated
from 100 sweeps, increased from 0.03 to 0.06 pA/ms.
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Similar effects of BCh on whole cell
Ca2+ channel currents were
measured using permeabilized-patch recordings (Fig.
6). In all five cells tested, BCh (100 µM) significantly increased the mean peak current from 100 ± 14 to 197 ± 27 pA. As shown in Fig. 6, the effect of BCh
was observed only at test potentials positive to
30 mV, with the
greatest increases in current occurring at test potentials positive to
20 mV.

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Fig. 6.
BCh increased whole cell Ca2+
currents in HIT-T15 cells. A: whole
cell Ca2+ currents evoked by
200-ms voltage steps (holding potential 70 mV) in absence
(top) and presence (bottom) of BCh (100 µM; 10 min). Test potentials ranged from 30 to +40 mV in 10-mV
increments. B: whole cell
current-voltage relationship in same cell determined using voltage
steps from 120 to +50 mV (holding potential 70 mV).
Exposure to 100 µM BCh for 10 min increased peak
Ba2+ current from 122 to 322 pA.
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Activation of PKC mimics BCh effects.
Because PKC has been implicated in muscarinic potentiation of
glucose-stimulated insulin secretion (16, 22, 23), we compared the
effects of exogenous activators of the enzyme with those of BCh. Acute
application of the active phorbol ester PMA (10-100 nM) resulted
in a marked increase in the frequency of Ca2+ channel openings (Fig.
7). Similar to the response to BCh, the increased channel activity was accompanied by significant
(P
0.05) increases in
Fo (0.03 ± 0.01 vs. 0.14 ± 0.02) and Iavg
(0.03 ± 0.01 vs. 0.14 ± 0.03 pA/ms) and a significant decrease
in
c2 (35.5 ± 1.1 vs. 11.1 ± 1.9 ms) in four of five patches tested. Unlike the response to
BCh, no significant increase in
o was observed. In contrast,
acute application of 4
-phorbol (100 nM), which does not activate
PKC, had no effect on Fo (0.05 ± 0.02 vs. 0.04 ± 0.01),
Iavg (0.04 ± 0.03 vs. 0.04 ± 0.01 pA/ms),
c2 (20.8 ± 6.0 vs. 20.6 ± 6.0 ms),
o (0.5 ± 0.1 vs. 0.6 ± 0.1 ms), or
c1
(1.4 ± 0.2 vs. 1.2 ± 0.2 ms) in all five patches tested. The
effects of the membrane-permeable DAG analog
DC10 on single Ca2+ channel currents were also
tested. Acute application of this compound also caused a significant
increase in Ca2+ channel activity
(Fig. 8) accompanied by significant
(P
0.05) increases in
Fo (0.04 ± 0.01 vs. 0.09 ± 0.01) and Iavg
(0.03 ± 0.01 vs. 0.10 ± 0.02 pA/ms) and a significant decrease
in
c2 (27.1 ± 3.8 vs. 12.8 ± 1.6 ms), but
o (0.4 ± 0.1 vs. 0.5 ± 0.1 ms) and
c1 (2.7 ± 0.7 vs. 1.7 ± 0.2 ms) were unchanged in six of eight patches tested.

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Fig. 7.
Phorbol ester phorbol 12-myristate 13-acetate (PMA) increased
high-threshold Ca2+ channel
currents in HIT-T15 cells. Top:
single-channel openings were evoked by voltage steps from a holding
potential of 70 mV to a test potential of 10 mV.
High-K+ bath solution contained
0.01% DMSO, which served as a vehicle control.
Bottom: 5 min after bath solution was
changed to one that contained PMA (100 nM; 0.01% DMSO),
Iavg, calculated
from 100 sweeps, increased from 0.02 to 0.06 pA/ms.
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Fig. 8.
Diacylglycerol analog
1,2-didecanoyl-rac-glycerol
(DC10) increased high-threshold
single Ca2+ channel currents in
HIT-T15 cells. Top: single-channel
currents were evoked by steps from a holding potential of 70 mV
to a test potential of 10 mV in a high (143 mM)-K+ bath solution containing
0.01% DMSO as a vehicle control.
Bottom: 5 min after bath solution was
changed to one containing DC10 (5 µg/ml; 0.01% DMSO),
Iavg, calculated
from 100 sweeps, increased from 0.04 to 0.11 pA/ms.
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Depletion or inhibition of PKC activity antagonizes effects of BCh.
To further test the role of PKC in muscarinic activation, we examined
the effects of depleting or inhibiting PKC activity. Depletion of PKC
activity was accomplished by chronic (20-32 h) exposure to PMA
(200 nM), a treatment known to deplete enzyme activity by >75% in
-cells (25). After such treatment, BCh caused only a small (20%)
but significant increase in Fo and
Iavg and no
change in the mean open or closed times in 9 of 12 cells tested. In
contrast, seven of nine cells chronically exposed to 4
-phorbol (200 nM) responded to BCh with large increases in
Fo (180%) and
Iavg (225%)
and a decrease (54%) in
c2
(Table 2). The nature and magnitude of
these effects were comparable to those observed previously in untreated
cells (Table 1).
We also examined the effects of BCh in the presence of the protein
kinase inhibitor staurosporine (100 nM). This concentration of
staurosporine has been reported to inhibit islet PKC activity by
>80% (12). Ca2+ channel
activity was initially recorded in the presence of staurosporine alone
before switching to a solution containing both staurosporine and BCh
(100 µM). Under these conditions, BCh evoked small but significant
increases in Fo (0.03 ± 0.01 vs. 0.04 ± 0.01) and Iavg (0.04 ± 0.01 vs. 0.05 ± 0.01 pA/ms), but no changes in
o (0.3 ± 0.1 vs. 0.3 ± 0.1 ms),
c1 (2.1 ± 0.3 vs.
1.9 ± 0.5 ms), or
c2 (24.6 ± 4.2 vs. 23.3 ± 5.9 ms) were observed in seven of nine cells
tested. In parallel control cells from the same passage, BCh, in the
presence of the vehicle (0.01% DMSO) alone, caused significant
increases in Fo (0.04 ± 0.01 vs. 0.10 ± 0.02) and Iavg (0.05 ± 0.01 vs. 0.10 ± 0.02 pA/ms) accompanied by a significant decrease in
c2 (15.9 ± 1.6 vs. 8.4 ± 2.2 ms), but
o
(0.3 ± 0.1 vs. 0.4 ± 0.1 ms) and
c1 (1.9 ± 0.6 vs. 1.2 ± 0.2 ms) were unchanged.
 |
DISCUSSION |
Ca2+ channels
in HIT-T15 cells.
HIT-T15 cells exhibited exclusively high-threshold voltage-dependent
Ca2+ channel currents. Both
single-channel and whole cell currents had thresholds of activation
positive to
40 mV that were unaltered when the holding potential
was increased from
60 to
100 mV. These high-threshold
currents were predominantly, if not exclusively, L-type currents.
Consistent with this conclusion were blockade of >80% of
single-channel and whole cell currents by nifedipine, a mean
single-channel conductance of 26 pS, little current inactivation during
prolonged depolarizations, and rapid, complete loss of single-channel
currents after excision of cell-attached patches. Using the solutions
and recording protocols that revealed low-threshold, T-type channels in
rat
-cells (3), we observed no such channels in cell-attached
patches from HIT cells. Our results are the first to characterize
single Ca2+ channel currents in
HIT-T15 cells and to correlate them with whole cell currents recorded
under conditions that preserved the normal cytosolic components
required for Ca2+ channel
activity.
Using standard whole cell recordings of HIT cell
Ca2+ currents, Keahey et al. (17)
reported thresholds between
50 and
40 mV from a holding
potential of
80 mV. These currents were reduced by
~85% in the presence of nimodipine, and the residual currents retained the kinetics of L-type
Ca2+ currents. Parallel studies of
the dihydropyridine sensitivity of
K+-stimulated insulin secretion
and intracellular Ca2+
accumulation revealed a strong correlation with that of the
Ca2+ currents. These
authors concluded that HIT cells contain almost exclusively
high-threshold, L-type Ca2+
channels and that, as reported in islets and primary cultures of
-cells, the entry of extracellular
Ca2+ essential for insulin
secretion occurs through these channels. Simultaneous measurements of
Ca2+ transients and whole cell
Ca2+ currents in HIT cells also
indicated that voltage-dependent influx was carried exclusively by
L-type channels (7). Our results are the first to confirm these
conclusions at the single-channel level.
Fast, Ca2+-dependent and slow,
voltage-dependent components of
Ca2+ current inactivation have
been described in HIT cells (28). Currents elicited at more negative
test potentials (
50 to
30 mV) were either unaffected or
increased by cell dialysis, whereas currents observed at more positive
potentials were subject to rundown, suggesting the presence of
high-threshold L- and N-type channels. An
-conotoxin
(CTX)-sensitive, high-threshold (N-type) Ca2+ current has also been
recorded in the rat insulinoma cell line RINm5F (30). This
high-threshold current comprised 15-25% of the total
Ca2+ current, and its blockade
with CTX inhibited stimulated insulin secretion by up to 51%. Although
our experiments cannot prove that such a current does not exist in HIT
cells, our results indicate that it could contribute only a small
fraction (<20%) of the total current, whereas in HIT cells over 90%
of insulin secretion is dihydropyridine sensitive (17). Thus the
effects of BCh and PKC activators on
Ca2+ currents in HIT cells can be
ascribed to actions on high-threshold, L-type channels.
Muscarinic agonist increased voltage-activated
Ca2+ channel
currents in HIT-T15 cells.
Stimulation of muscarinic receptors increased single-channel and whole
cell L-type Ca2+ channel currents
two- to threefold in HIT-T15 cells under recording conditions that
chemically or electrically clamped resting membrane potential. This is
the first direct evidence for a muscarinic receptor-mediated modulation
of single Ca2+ channel currents in
pancreatic
-cells that is independent of effects on resting membrane
potential. Antagonism of the BCh effects by atropine confirmed that
this was a muscarinic receptor-mediated event, and the PTX-resistant
nature of the effects suggests coupling of the receptor to G proteins
other than Gi or
Go, which are associated with
PTX-sensitive inhibition of L-type
Ca2+ channels in
-cells (29).
These observations are consistent with previous reports that
cholinergic potentiation of glucose-stimulated insulin secretion is
mediated by PTX-resistant G proteins (30, 34). Recent work indicates
that muscarinic M3 receptors
mediate cholinergic potentiation of glucose-stimulated insulin
secretion (5), and in other tissues, these receptors appear
preferentially coupled to Gq and
G11 and lead to the activation of
phospholipase C
1 (18). The specific stimulatory G proteins mediating
cholinergic activation of phospholipase C in islets remain to be
determined; however, one recent study has implicated
Go (33).
Muscarinic stimulation of
Ca2+ currents
required PKC activation.
Cholinergic agonists activate phospholipase C with the production of
IP3 and DAG in both pancreatic
islet cells and HIT cells. These two second messengers respectively
cause the release of intracellular
Ca2+ and the activation of PKC (8,
15, 16, 20, 22, 23, 34, 36). In HIT-T15 cells activation of PKC, using
either a phorbol ester or a DAG analog, qualitatively and
quantitatively mimicked the effects of BCh on single and whole cell
Ca2+ channel currents. Conversely,
both the PKC inhibitor staurosporine and depletion of PKC activity by
chronic phorbol treatment markedly inhibited the effects of BCh.
Biochemical studies of islet and HIT cells have demonstrated that
staurosporine, at the concentrations used in our experiments, inhibited
PKC activity by 70-80% (37), whereas chronic exposure to phorbol
esters reduced PKC activity by 75%. These effects were accompanied by
a comparable inhibition of the sustained cholinergic potentiation of
glucose-stimulated insulin secretion that was dependent on
Ca2+ influx (16, 22, 23, 32). We
observed a comparable inhibition of BCh-stimulated increases in
Ca2+ currents. Thus intact PKC
activity appears essential for the muscarinic effects that we observed.
These results do not preclude the possibility that PKC might activate
another second-messenger system, which in turn modulates Ca2+ channels. cAMP has been shown
to increase Ca2+ influx through
voltage-dependent channels in
-cells (1, 24), and we have also
recorded significant (P
0.05)
increases in Fo (0.04 ± 0.01 vs. 0.10 ± 0.01; 5 of 6 cells) of single
Ca2+ channel currents in HIT-T15
cells exposed to forskolin (5-10 µM) in the presence of
3-isobutyl-1-methylxanthine (100 µM). The membrane-permeable analog
8-bromo-cAMP (2 mM) also increased
Fo (0.05 ± 0.01 vs. 0.09 ± 0.03; 3 of 3 cells) in HIT-T15 cells (unpublished observations).
However, numerous biochemical studies on islet cells and clonal
-cell lines have demonstrated muscarinic potentiation of
glucose-stimulated insulin secretion without any effect on cAMP levels
(9, 20, 36). The fact that our experiments were conducted with the
cells depolarized to a membrane potential at or near 0 mV in the
presence of extracellular Ca2+
argues against the activation of another protein kinase by BCh due to
increased Ca2+ influx. Our
experiments also cannot rule out some direct modulation of
Ca2+ channel activity by
interaction with G protein subunits.
Potential importance in insulin secretion.
Numerous studies have confirmed that cholinergic agonists produce a
biphasic potentiation of glucose-stimulated insulin secretion. An
initial rapid, brief increase in secretion, mediated by
IP3 generation and release of
intracellular Ca2+, is followed by
a larger, prolonged PKC-mediated increase in secretion that is
dependent on extracellular Ca2+
(11, 15, 16, 20, 22, 23, 25, 32, 34, 36). Previous work indicated that
cholinergic agonists, in the presence of substimulatory concentrations
of glucose, depolarized the membrane potential of
-cells (6, 9, 20,
27) and increased Na+ influx (12)
but potentiated cyclic electrical activity,
Ca2+ influx, or insulin secretion
only in the presence of ongoing electrical activity initiated by
glucose or some other depolarizing secretagogue (9, 11, 14, 16, 20, 26,
36). To date, two mechanisms have been postulated for the PKC-mediated
effects: an increase in Ca2+
influx secondary to membrane depolarization, and a sensitization of the
secretory process to Ca2+ (6, 9,
11-16, 22, 23, 26, 27). Our results suggest a third, PKC-mediated,
cholinergic mechanism, an increase in
Ca2+ channel activity at any given
membrane potential above the threshold for channel opening. Given the
close correlation of intracellular Ca2+ concentration and insulin
secretion (20, 36), an increase in the
Ca2+ currents would be expected to
contribute to increased insulin secretion. It is likely that all three
mechanisms, membrane depolarization, direct
Ca2+ channel activation, and
sensitization of exocytosis, act in concert during cholinergic
potentiation of glucose-stimulated insulin secretion.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-44169, the University of
Michigan Diabetes Research and Training Center, and the University of
Michigan Gastrointestinal Peptide Research Center.
 |
FOOTNOTES |
Address for reprint requests: J. A. Love, Dept. of Pharmacology and
Toxicology, University of Mississippi Medical Center, 2500 North State
St., Jackson, MS 39216-4505.
Received 26 March 1997; accepted in final form 28 October 1997.
 |
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