(Received for publication, November 1, 1994; and in revised form, March 27, 1995)
From the
Glucagon-like peptide-1 (GLP-1) is an intestinally derived
insulinotropic hormone that is currently under investigation for use in
the treatment of diabetes mellitus. To investigate the Ca GLP-1 These insulinotropic actions prompted interest in the potential
therapeutic use of GLP-1 as an antidiabetogenic (blood
glucose-lowering) agent. Intravenously administered GLP-1 raises
circulating levels of insulin and lowers blood glucose in
non-insulin-dependent
diabetics(16, 17, 18, 19) . GLP-1
also offers several distinct advantages over sulfonylureas in the
treatment of non-insulin-dependent diabetes. It exerts a stronger
antidiabetogenic effect than is achieved following oral administration
of glyburide, and it is effective even under conditions in which
glyburide fails to control blood glucose levels
adequately(18) , a condition referred to as sulfonylurea
failure(21) . Unlike sulfonylureas, the antidiabetogenic effect
of GLP-1 is glucose-dependent. GLP-1 fails to stimulate insulin
secretion when blood glucose levels fall to below low normoglycaemic
levels, approximately 5
mM(1, 2, 3, 7) . Therefore,
the action of GLP-1 to stimulate insulin secretion and to lower blood
glucose is self-correcting, and the risk of hypoglycemia as an untoward
side effect of GLP-1 treatment is reduced greatly. The mechanisms by
which GLP-1 potentiates glucose-induced insulin secretion from
pancreatic GLP-1 stimulates the production of
cAMP, a second messenger that has important effects on To further investigations of how GLP-1 influences
insulin secretion, we examined its effects on
[Ca
where K
Test solutions containing GLP-1, pituitary
adenylyl cyclase-activating peptide-27 (PACAP-27), glucagon, forskolin,
Sp- and Rp-cAMP-S, IBMX, or 8-Br-cAMP, were applied to individual cells
by focal application from ``puffer'' micropipettes (26) using a PicoSpritzer II pressure ejection system (General
Valve, Fairfield, NJ). For experiments examining the effects of
peptides, the test solutions also contained 0.05% human serum albumin
(fraction V; Sigma) added to protect against absorption of the peptides
to borosilicate glass culture tubes in which the solutions were
prepared. Human serum albumin was found to be without effect on the
measurements reported here. A superfusion system driven by a Ismatec
direct current-powered peristaltic pump (Cole-Palmer Instrument Co.,
Chicago) applied known concentrations of Ca GLP-1(7-37) was obtained from Peninsula Laboratories (Belmont,
CA). GLP-1(7-36)amide, PACAP-27, glucagon, forskolin, 8-Br-cAMP,
IBMX, nifedipine, nimodipine, verapamil, and diazoxide were obtained
from Sigma. Sp- and Rp-cAMP-S were obtained from BioLog Life Sciences
Institute (Bremen, FRG). Tetrodotoxin and ryanodine were obtained from
Calbiochem.
The patch pipette was
connected to an Heka Electronik EPC-9 patch clamp amplifier (Instrutech
Corp., Mineola, NY) interfaced with a Macintosh Quadra 840AV computer
running Pulse software (Instrutech Corp.). The series resistance (R
Figure 1:
GLP-1 increases
[Ca
In
Figure 2:
Effects of ion substitution on GLP-1
binding and cAMP production. Panel A, omission of
Na
In contrast, omission of extracellular
Na
Figure 3:
The
rise of [Ca
Figure 4:
The rise of
[Ca
The rise of
[Ca
Figure 5:
Effects of GLP-1 on membrane potential (V). Panel A, illustrated is a perforated patch
current clamp measurement of membrane potential obtained from a rat
Simultaneous measurements of membrane potential and
[Ca
Figure 6:
Simultaneous measurements of membrane
potential and [Ca
Application of 8-Br-cAMP to
Figure 7:
Activation of I
I An estimate of the
reversal potential for the GLP-1-induced inward current was determined
for a rat Measurements obtained from a voltage-clamped HIT-T15 cell
demonstrated that 8-Br-cAMP also induced an inward current and an
increased membrane conductance (Fig. 8A). This was
accompanied by a large rise of
[Ca
Figure 8:
I
First,
I The two isopeptides of GLP-1, namely GLP-1(7-37) and
GLP-1(7-36)amide, have attracted considerable attention because
of their ability to stimulate insulin gene expression and proinsulin
biosynthesis and to potentiate glucose-induced insulin
secretion(1, 7) . Since these actions of GLP-1 are
known to be glucose-dependent (1, 2, 3) and
since GLP-1 is a potent stimulator of cAMP
production(8, 14, 22, 23, 24, 25, 31, 53) ,
it has been proposed that GLP-1 is a modulator of the pancreatic
The ability of GLP-1 to raise
[Ca Fig. 9provides a model with which to evaluate these diverse
actions of GLP-1. Receptor occupancy by GLP-1 activates G
Figure 9:
Signaling pathways that may mediate the
stimulatory effects of GLP-1 on [Ca
A distinguishing feature of
the GLP-1 signaling system is its absolute dependence on extracellular
Na We also find that omission of extracellular
Na The
exact mechanism by which GLP-1 activates I As indicated above, Ca-NS channels that are activated by cAMP and
which are permeant to both Na The activation of I A clue as to the nature of this voltage-independent
process is provided by previous studies demonstrating that nonselective
cation channels can allow permeation by Ca It is important to point out that our findings do not rule out the
additional possibility that GLP-1 and cAMP raise
[Ca From a functional standpoint, the ability of GLP-1 to raise
[Ca
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
signaling pathways by which GLP-1 may stimulate the secretion of
insulin from pancreatic
-cells, we examined its effects on the
concentration of free intracellular Ca
([Ca
]
) while
simultaneously determining what action it exerts on ion channel
function. Measurements of [Ca
]
were obtained from single rat
-cells and from
TC6
and HIT-T15 insulinoma cells loaded with the Ca
indicator fura-2, and changes in membrane potential and current
were monitored using the perforated patch clamp technique. We report a
previously undocumented action of GLP-1 and analogs of cAMP
(8-bromo-cAMP, Sp- or Rp-adenosine 3`,5`-cyclic monophosphothionate
triethylamine) to raise [Ca
]
that is attributable to the activation of a prolonged inward
current designated here as I
. Activation of I
is associated with an increased membrane conductance, membrane
depolarization, and triggers large increases of
[Ca
]
. I
is primarily a Na
current that is blocked by
extracellularly applied La
or by intracellular
administration of Ca
chelators
(1,2-bis(2aminophenoxy)ethane-N,N,N`,N`-tetraacetic
acid/acetoxymethyl, EGTA) and which exhibits a reversal potential of
about -26 mV. We propose that I
results from the
opening of nonselective cation channels that are activated by
intracellular Ca
and cAMP and which might play an
important role in the regulation of insulin secretion from pancreatic
-cells.
(
)is an intestinally derived
hormone that plays an important role in systemic glucose homeostasis (1, 2, 3) . GLP-1 is derived by
tissue-specific post-translational processing of proglucagon (4, 5) and is secreted from enteroendocrine cells
(L-cells) of the intestinal tract in response to a meal or an oral
glucose challenge(1, 2, 3) . GLP-1 is a
gluco-incretin hormone because it mediates humoral communication among
the intestine, the pancreas, and target sites of insulin action (the
entero-insular axis), thereby exerting important influences on glucose
uptake and disposal (1, 2, 3, 6, 7) . GLP-1 is
also an insulinotropic hormone by virtue of its ability to stimulate
insulin gene expression(8, 9) , proinsulin
biosynthesis(9) , and to potentiate glucose-induced insulin
secretion from pancreatic
-cells(10, 11, 12, 13, 14, 15) .
-cells have yet to be elucidated fully. The active
forms of GLP-1 in vivo are the isopeptides GLP-1(7-37)
and GLP-1(7-36)amide, both of which bind to G protein-coupled
receptors on
-cells(22, 23, 24) , and
which stimulate production of
cAMP(8, 14, 22, 23, 24, 25) .
GLP-1 also enhances the inhibitory effect of glucose on the
sulfonylurea-sensitive potassium current (I
ATP), thereby
augmenting glucose-induced membrane depolarization(26) . This
synergistic interaction results in activation of voltage-dependent
calcium channels (VDCCs), thereby raising
[Ca
]
(26, 27) .
Since a rise in [Ca
]
triggers insulin secretion (28, 29) and
because GLP-1 increases [Ca
]
in
-cells (30, 31, 32) , such
a sequence of events may contribute to the reported effects of GLP-1 on
stimulus-secretion coupling.
-cell
function. These effects include augmentation of electrical
activity(26, 33, 34, 35) , membrane
depolarization(26, 34, 35) , Ca
influx(35, 36, 37, 38, 39) ,
and insulin
secretion(34, 35, 36, 37, 40, 41) .
GLP-1 also increases [Ca
]
and potentiates glucose-induced insulin secretion in a
Na
-dependent manner(32, 42) , a
requirement that suggests the involvement of a Na
permeability change as an underlying feature of the action of
GLP-1. In contrast, cAMP activates nonselective cation channels in the
CRI-G1 insulinoma cell line(43) , stimulates
Ca
efflux from secretory granules in
-cells(44) , and augments insulin secretion by
facilitating fusion of secretory granules with the plasma
membrane(45, 46) . Therefore, no single mechanism of
action accounts for the stimulatory effects of GLP-1 and cAMP in the
-cell system.
]
while also
determining what action it exerts on ion channel function. Measurements
were obtained from rat
-cells maintained in short term primary
cell culture. Also studied were
TC6 and HIT-T15 insulinoma cells,
two cell lines that secrete insulin in response to glucose (47, 48, 49, 50, 51) and
which express GLP-1 receptors that couple to cAMP
production(8, 14, 31, 53) . We
report that GLP-1 and analogs of cAMP raise
[Ca
]
by activating an
inward current designated here as I
. Activation of
I
is accompanied by an increased membrane conductance,
membrane depolarization, and large increases of
[Ca
]
which are
dependent on extracellular Ca
and
Na
. These findings document a novel GLP-1-sensitive
Ca
-signaling pathway that might play an important
role in the regulation of insulin secretion from
-cells.
Preparation of Cell Cultures
HIT-T15
cells were obtained from the American Type Culture Collection. TC6
cells were obtained from Dr. Shimon Efrat (Albert Einstein College of
Medicine, New York). HIT-T15 cells were maintained in Ham's F-12
medium containing 10 mM glucose, 10% heat-inactivated horse
serum, and 2.5% fetal bovine serum.
TC6 cells were maintained in
Dulbecco's modified Eagle's medium containing 25 mM glucose, 15% horse serum, and 2.5% fetal bovine serum. Culture
media also contained 100 units/ml penicillin G and 100 µg/ml
streptomycin. Primary cultures of rat
-cells were prepared and
maintained as described previously(26) . Cells were plated onto
glass coverslips coated with 1 mg/ml type V concanavalin A (Sigma),
which facilitates their adherence to glass. Cultures were maintained at
37 °C in a 5% CO
atmosphere incubator, and experiments
were conducted 1-5 days postplating.
Measurement of Intracellular
Calcium
Cells were prepared for measurement of
[Ca]
by incubation in
fura-2 acetoxymethyl ester (fura-2/AM; Molecular Probes, Inc., Eugene,
OR). Cells were loaded in saline containing 2% fetal bovine serum,
0.03% pluronic F-127, and 0.5-5 µM fura-2/AM for
10-30 min at 20-22 °C. Under these conditions, 91% of
the fluorescence emission is attributable to cytosolic
fura-2(54) . Coverslips with fura-loaded adherent cells formed
the base of a recording chamber mounted on a temperature-controlled
stage (MicroDevices, Jenkintown, PA). Cells were visualized using a
Zeiss IM35 microscope equipped with a Nikon UVF100 100X objective.
Measurements of [Ca
]
were performed at 1-s intervals using a dual excitation
wavelength video imaging system (IonOptix Corp., Milton, MA).
Experiments were conducted at 32 °C.
[Ca
]
was estimated
from the ratio of 510 nm emission fluorescences due to excitation by
350 nm and 380 nm wavelength light from (55)
is the dissociation
constant of fura-2 (225 nM),
is the ratio of 380 nm
induced fluorescences of free/bound fura-2, R is the measured
ratio of 350 nm/380 nm fluorescences, and R
and R
are 350 nm/380 nm fluorescence ratios in zero
[Ca
] and saturating
[Ca
], respectively. Values of
, R
, and R
were determined
using fura-2 pentapotassium salt and calibration solutions from
Molecular Probes, Inc.
Preparation of Test Solutions
Cells for
fura-2 loading and patch clamp recording were bathed in a standard
extracellular buffered saline containing 138 mM NaCl, 5.6
mM KCl, 2.6 mM CaCl, 1.2 mM MgCl
, 10 mM HEPES (295 mosm; pH adjusted to
7.4 with NaOH). The concentration of D-glucose was adjusted to
be near threshold for stimulation of insulin secretion (7.5 mM for rat
-cells(28) , 0.8 mM for insulinoma
cells(47, 58) ). Na
-free solutions
were prepared using 138 mMN-methyl-D-glucamine (NMG) substituted for NaCl/NaOH
and adjusted to pH 7.4 with HCl. In some experiments 138 mM Tris-HCl (pH 7.4) was used as a substitute for Na
in place of NMG. Solutions to which no Ca
was
added were prepared by substituting MgCl
for
CaCl
.
and
Na
channel blockers to the superfusate.
-Conotoxin GVIA was from Bachem California (Torrance,
CA).
Patch Clamp Recording Techniques
The
resting potential and holding current were measured under current clamp
or voltage clamp using the tight seal, whole cell, perforated patch
configuration(56, 57) . Patch pipettes pulled from
borosilicate glass (Kimax-51, tip resistance 2-3 megohms) were
fire polished and tip dipped in 95 mM KSO
, 7 mM MgCl
, 5
mM HEPES (300 mosm; pH adjusted to 7.4 with NaOH; final
concentration of Na
equal to about 2 mM), and
back filled with the same solution containing nystatin (240 µg/ml).
A limited number of experiments utilized the standard whole cell
recording configuration in which the patch membrane was ruptured, and
diffusional exchange was allowed to occur between the pipette solution
and the cytosol. Under these conditions the pipette solution contained
either a ``Ca
-free'' intracellular solution
containing (in mM): 140 KCl, 2 mM MgCl
,
10 HEPES/KOH (pH 7.4), and 5 EGTA, or an intracellular solution
containing 160 nM Ca
and composed of (in
mM): 140 KCl, 2 mM MgCl
, 10 HEPES/KOH (pH
7.4), 0.1 CaCl
, and 1.1 EGTA.
) and cell capacitance (C
)
were monitored following seal formation, and experiments were conducted
when R
declined to 12-25 megohms and C
increased to 10-40 picofarads. Electrical
access was confirmed by noting a -50 to -70 mV resting
potential and by noting a rise in
[Ca
]
in response to a
depolarizing voltage step. The experiment was rejected if such a
[Ca
]
response was not
noted. In voltage clamp experiments, R
was
compensated for by 60-80%. The pipette solutions did not contain
fura-2 and were nominally Ca
-free unless otherwise
noted. A sudden decrease in R
accompanied by a
rise in [Ca
]
and
decrease in fluorescence intensity provided a useful marker for
unintended rupture of the cell membrane during perforated patch
recordings, whereupon the experiment was terminated.
GLP-1 Receptor Binding Assays
The
specific binding of I-GLP(7-37) or
I-GLP(7-36)amide to the GLP-1 receptor was
determined by a rapid filtration binding assay in which the binding of
ligand to receptor was monitored using intact COS-7 cells transiently
transfected by the DEAE-dextran method with the rat GLP-1 receptor cDNA
encoded within the pcDNA-1 expression vector(22) .
I-GLP(7-37) and
I-GLP(7-36)amide were prepared by the chloramine-T
method of iodination, and the monoiodinated form of the peptide was
purified by high performance liquid chromatography. Bound radioligand
was separated from unbound by filtration through Whatman GF/C filters
pretreated with Krebs bicarbonate buffer containing 6% fetal bovine
serum and 0.8% Tween 20. Total binding was determined after
equilibration of cells with iodinated peptide for 60 min at 22 °C
in HEPES-buffered saline containing 2% human serum albumin. Nonspecific
binding was defined as binding of the radioligand observed following
pretreatment of cells with a 1 µM concentration of the
corresponding nonradioactive peptide. Specific binding was calculated
as the difference between total and nonspecific binding. Using this
approach,
1% of the radioligand bound nonspecifically, and
10-20% bound specifically.
Radioimmunoassay for cAMP
TC6 cells
were cultured in 24-well tissue culture plates until reaching
70-80% confluence. Extracellular solution (with 138 mM
Na
or 138 mM NMG substituted for
Na
) with 100 µM IBMX ± 10 nM GLP-1 (a concentration shown previously (53) to be
saturating for cAMP production in
TC1 cells) was substituted for
the culture medium, and the exposure to GLP-1 was allowed to progress
for 30 min at room temperature. Ice-cold absolute ethanol (1 ml) was
then added to each well, and the cells were subjected to three rounds
of freeze-thawing. The lysed cells and extracellular solution were
collected, and the total content of cAMP was measured by a specific
radioimmunoassay as described previously(53) .
GLP-1 Induces a Sustained Rise of
[Ca
To determine in what manner GLP-1
influences cytosolic Ca]
Which Is
Glucose-dependent
levels, measurements of
[Ca
]
were obtained
from single rat
-cells and insulinoma cells equilibrated in buffer
containing 7.5 and 0.8 mMD-glucose, respectively.
Under these conditions the cells exhibited stable membrane potentials
(-62 ± 7 mV; mean ± S.D.; n = 10
TC6 cells) and stable, low levels of
[Ca
]
(91 ± 8
nM; n = 50
TC6 cells). An increase of
[Ca
]
was observed in
response to a 10-s focal application of GLP-1(7-37) to single rat
-cells (Fig. 1A, n = 20 cells),
TC6 cells (Fig. 1B, n = 40), and
HIT-T15 cells (Fig. 1C; n = 20). The
sustained nature of these responses is not explained by the kinetics of
the drug delivery system used (26) since the superfusion system
ensures that GLP-1 is eliminated from the solution surrounding the
cells within 30 s. An increase of
[Ca
]
was also observed
in response to GLP-1(7-36)amide, not only in
TC6 cells (Fig. 1D; n = 20) but also in rat
-cells (n = 20) and HIT-T15 cells (n = 20). These responses were variable with respect to
reversibility. In some cells
[Ca
]
recovered to
prestimulus levels (Fig. 1, A and B), whereas
in others the [Ca
]
exhibited only partial recovery (Fig 1, C and D). No consistent differences were noted in the amplitude,
kinetics, or reversibility of responses when comparing effects of
GLP-1(7-37) versus GLP-1(7-36)amide.
] in rat
-cells and pancreatic
insulinoma cells. Each of the two isoforms of GLP-1, abbreviated as (7-37) or (7-36)a, were administered to
individual cells at a concentration of 10 nM for 10 s (arrows indicate the start of application). The GLP-1 was then
removed within 30 s by a superfusion system. A sustained rise of
[Ca
] in response to GLP-1 was observed in a
rat
-cell maintained in primary cell culture (panel A),
in
TC6 cells (panels B and D), and an HIT-T15
cell (panel C). In panel A the extracellular solution
contained 7.5 mM glucose, whereas in panels B-D it contained 0.8 mM glucose.
TC6 cells the GLP-1(7-37)-induced rise of
[Ca
]
was
dose-dependent and was diminished by removal of extracellular D-glucose (Table 1). These responses also exhibited
desensitization (Fig. 1B) to repeated application of
concentrations of the peptides (
10 nM) reported to induce
homologous desensitization of GLP-1-induced insulin secretion (53) . The action of GLP-1 to raise
[Ca
]
was mimicked by
activators of cAMP signaling pathways, including PACAP-27, glucagon,
forskolin, IBMX, Sp-cAMP-S, and 8-Br-cAMP (Table 2). Notably, the
cAMP antagonist Rp-cAMP-S was an effective agonist in this system (Table 2).
The GLP-1-induced Rise of
[Ca
The GLP-1-(7-37)-induced rise of
[Ca]
Requires Extracellular
Na
]
in
TC6 cells
was blocked by omission of Na
from the extracellular
bathing and test solutions (Table 3). This was observed when
Na
was replaced by either NMG or Tris-HCl. The failure
of GLP-1 to raise [Ca
]
in the absence of Na
was not due to the
inability of GLP-1 to bind to its receptor. Radioligand binding assays
using
I-GLP-1(7-37)
or(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) amide
confirmed that the binding of the peptides to the rat islet GLP-1
receptor expressed in transfected COS-7 cells was unaffected by
omission of Na
from the extracellular solution (Fig. 2A).
or Ca
from the extracellular
solution had no effect on the binding of GLP-1 to its receptor. COS-7
cells were transiently transfected with the rat islet GLP-1 receptor
cDNA, and the binding of
I-GLP-1(7-37) was
determined 3 days post-transfection. Illustrated are displacement
curves generated under conditions in which the binding of
I-GLP-1(7-37) was competed for by prior
equilibration of cells with the indicated concentration of
nonradioactive GLP-1(7-37). Under control conditions in which the
extracellular solution contained 138 mM Na
and 2.6 mM Ca
, approximately 20% of
added tracer was bound specifically, and the IC
for
displacement of radioligand by nonradioactive peptide was determined to
be approximately 2 nM. Neither Na
omission
(by replacement of Na
with NMG) nor Ca
omission (by replacement of Ca
with
Mg
) influenced either the total binding activity or
the IC
value for displacement of radioligand. Nearly
identical results were obtained using
I-GLP-1(7-36)amide as radioligand and
nonradioactive GLP-1(7-36)amide as displacer (data not shown). Panel B, the stimulation of
TC6 cell cAMP production by
GLP-1 was dependent on extracellular Na
. Cells were
incubated without (Control) or with GLP-1 for 30 min at 23
°C in extracellular solution containing 0.8 mM glucose,
100 µM IBMX, 2.6 mM CaCl
, and either
138 mM NaCl or 138 mM NMG (no Na
).
Total cAMP production was determined by radioimmunoassay. Statistical
significance relative to the control was evaluated by the t test (*, p
0.05;**, p
0.01).
markedly attenuated GLP-1-induced production of
cAMP in
TC6 cells (Fig. 2B). This observation
suggested that the failure of GLP-1 to influence
[Ca
]
under conditions
of Na
omission might be simply attributed to its
failure to generate cAMP, a requisite second messenger. It was noted,
however, that omission of extracellular Na
also
blocked the ability of forskolin (Fig. 3A) and
8-Br-cAMP (Fig. 3B) to increase
[Ca
]
in
TC6
cells. These findings suggested that Na
is an
obligatory cation, acting to influence multiple steps in the GLP-1
signaling system.
] evoked by forskolin and
8-Br-cAMP requires extracellular Na
. Illustrated in panels A and B are measurements of
[Ca
] obtained from
TC6 cells
equilibrated in Na
-free extracellular solution that
contained 0.8 mM glucose and 138 mM NMG substituted
for Na
. Focal application for 30 s of extracellular
solution containing 138 mM Na
had no effect
on [Ca
] (panels A and B).
Similarly, [Ca
] was not influenced by a
30-s application of Na
-free extracellular solution
containing 10 µM forskolin (panel A) or 1 mM 8-Br-cAMP (panel B). In contrast, an increase of
[Ca
] was observed in response to
simultaneous application of Na
and forskolin (panel A) or Na
and 8-Br-cAMP (panel
B). Data presented are representative of findings obtained in five
experiments using 20 cells (n = 5 forskolin; n = 15 8-Br-cAMP).
The GLP-1 and 8-Br-cAMP-induced Rise of
[Ca
The response of ]
Also Requires
Extracellular Ca
TC6
cells to GLP-1 could not be initiated under conditions in which
Ca
was omitted from the extracellular bathing and
test solutions (Table 3). Moreover, when
TC6 cells were
bathed in a solution containing Ca
, no response was
observed when the cells were challenged for 10 s with a
Ca
-free test solution containing 8-Br-cAMP and 50
µM EGTA, whereas a large rise of
[Ca
]
was observed upon
exposure to a test solution containing Ca
and
8-Br-cAMP (Fig. 4A). Furthermore, the rise of
[Ca
]
observed after
treatment with GLP-1 or 8-Br-cAMP was reversed within 1 min following
superfusion of the cells with a nominally Ca
-free
bath solution (Fig. 4B). This requirement for
Ca
was observed even though the binding of
I-GLP-1(7-37) to its receptor appeared normal under
conditions of the omission of extracellular Ca
(Fig. 2A).
] evoked by 8-Br-cAMP requires
extracellular Ca
and is not blocked by nimodipine.
Illustrated in panels A-C are measurements of
[Ca
] obtained from
TC6 cells
equilibrated in the standard extracellular solution containing
Na
, Ca
, and 0.8 mM glucose. Panel A, the cell failed to respond to repeated 10-s
applications of 1 mM 8-Br-cAMP dissolved in a
Ca
-free extracellular solution containing 50
µM EGTA. A subsequent 10-s application of 1 mM 8-Br-cAMP dissolved in the standard extracellular solution
containing Ca
resulted in a large increase of
[Ca
]. Panel B, the cell was
challenged with a 10-s application of 1 mM 8-Br-cAMP dissolved
in the standard extracellular solution containing Ca
.
A large increase of [Ca
] was observed which
was reversed by equilibration of the cell in a nominally
Ca
-free extracellular solution introduced into the
recording chamber by superfusion. Panel C, a 10-s application
of a depolarizing concentration (56 mM) of KCl resulted in a
large rise of [Ca
] due to activation of
VDCCs. Bath application of 1 µM nimodipine substantially
reduced the rise of [Ca
] evoked by a test
solution containing 56 mM KCl and 1 µM nimodipine
applied for 10, 20, and 30 s, as indicated. A large rise in
[Ca
] was then observed in response to a
10-s application of a test solution containing 1 mM 8-Br-cAMP
and 1 µM nimodipine.
Pharmacological Characterization of the GLP-1-induced
Rise of [Ca
In
]
TC6 cells the response to GLP-1 (and 8-Br-cAMP; n = 5; data not shown) was abrogated by extracellular
application of 10 µM La
(Table 4),
a broad spectrum antagonist of VDCCs which at this concentration also
blocks nonselective cation channels. In contrast, nifedipine and
verapamil, two specific antagonists of L-type VDCCs, did not block the
response to GLP-1 in either cell type, although a statistically
significant inhibitory trend was noted (Table 4). As was the case
for GLP-1, the rise of [Ca
]
in response to 8-Br-cAMP was also not blocked by the L-type
VDCC antagonist nimodipine, whereas the rise of
[Ca
]
observed during
exposure to a depolarizing concentration (56 mM) of KCl was
markedly attenuated (Fig. 4C).
]
in response to
GLP-1 (and 8-Br-cAMP; n = 5; data not shown) was
significantly reduced but not blocked by diazoxide, an activator of
I
ATP which chemically clamps the
-cell membrane
potential to a value close to the K
equilibrium
potential, thereby preventing activation of VDCCs (Table 4). In
contrast, the response to GLP-1 was not blocked by
-conotoxin
GVIA, a blocker of N-type VDCCs, nor was it eliminated by ryanodine, a
blocker of intracellular Ca
release channels (Table 4). Furthermore, tetrodotoxin, a blocker of
voltage-dependent Na
channels, was also without effect (Table 4).
Effects of GLP-1 and 8-Br-cAMP on Membrane
Potential
Perforated patch clamp measurements obtained from
rat -cells equilibrated in a steady-state concentration of glucose
(7.5 mM) revealed that a 10-s exposure to GLP-1 produced a
prolonged and reversible depolarizing shift in membrane potential from
an initial value of about -60 mV to a plateau value of ca.
-10 mV (Fig. 5A, n = 10).
Membrane depolarization in response to GLP-1 was also observed in
HIT-T15 cells (Fig. 5B, n = 10) and
TC6 cells (n = 5; data not shown). The onset of
the depolarization was accompanied by the generation of numerous action
potentials, as evidenced by spike-like phenomena on the rising phase of
the response (Fig. 5, A and B).
-cell equilibrated in buffer containing 7.5 mM glucose.
Application for 10 s of 10 nM GLP-1(7-37) produced
sustained membrane depolarization and the generation of action
potentials (spike-like phenomena). Panel B, illustrated are
the sustained membrane depolarization and generation of action
potentials observed in response to 10 nM GLP-1(7-36)amide applied for 10 s to an HIT-T15 cell
equilibrated in buffer containing 0.8 mM glucose.
]
obtained from an
HIT-T15 cell demonstrated that a rise of
[Ca
]
was associated
with the increased excitability and membrane depolarization observed
during exposure to GLP-1 (Fig. 6A). In voltage-clamped
cells, a rise of [Ca
]
was also observed in response to a stepwise shift of the
membrane potential from -70 to 0 mV for 10 s, thereby confirming
the presence of VDCCs in this cell type (Fig. 6A). Such
responses to GLP-1 were also obtained in rat
-cells (n = 5) and
TC6 cells (n = 5).
]. Panel A, a
10-s application of 10 nM GLP-1(7-37) to an HIT-T15 cell
equilibrated in 0.8 mM glucose resulted in membrane
depolarization and the generation of action potentials (top
trace) accompanied by rise of [Ca
] (bottom trace). The cell was then placed under voltage clamp
(indicated by an arrow in the top trace), and the
membrane potential was held at -70 mV. A stepwise shift of the
membrane potential from -70 to 0 mV for 10 s produced a large
rise of [Ca
]. Panel B, application
of 1 mM 8-Br-cAMP for 10 s to a
TC6 cell equilibrated in
0.8 mM glucose produced membrane depolarization and the
generation of action potentials (top trace) and a rise of
[Ca
] (bottom trace). The
8-Br-cAMP-induced rise of [Ca
] was reduced
by voltage clamping the membrane to -70 mV and was augmented by
stepping the membrane potential from -70 to -100
mV.
TC6 cells also resulted in membrane
depolarization and a rise of [Ca
]
to a new steady-state level (Fig. 6B; n = 5). Voltage clamping the membrane to -70 mV at the
peak of the response resulted in a rapid reduction of
[Ca
]
, as expected if
the initial rise of [Ca
]
resulted, at least in part, from the opening of VDCCs. A
subsequent shift of the membrane potential from -70 to -100
mV resulted in a large increment of
[Ca
]
, suggesting an
additional mechanism of Ca
entry distinct from VDCCs (Fig. 6B). Similar responses were also observed in rat
-cells (n = 5) and HIT-T15 cells (n = 10), not only in response to 8-Br-cAMP but also in
response to GLP-1 (n = 5 HIT-T15 cells).
Activation of I
Measurements obtained from voltage-clamped
by GLP-1 and
8-Br-cAMP
TC6 cells revealed that the actions of GLP-1 were associated with
the appearance of I
indicated by an inward shift of the
holding current (I
, Fig. 7A, trace
1). I
outlasted the transient (10-s) application of
the peptide and was accompanied by a gradual rise of
[Ca
]
(Fig. 7A, trace 2). Such inward
current responses (typically 200-400 pA; -70 mV holding
potential) were observed in 10/10
TC6, 7/10 HIT-T15, and 4/5 rat
-cells tested.
and a rise
of [Ca
] by GLP-1(7-37). Panel
A, simultaneous measurements of membrane current (trace
1) and [Ca
] (trace 2) were
obtained from a
TC6 cell equilibrated in 0.8 mM glucose.
I
was monitored while voltage clamping the membrane at
-70 mV in the perforated patch configuration. Measurements of
membrane conductance were obtained by applying ±10-mV shifts in
the holding potential at 5-s intervals. These shifts in membrane
potential evoked outward (I) and inward (I) current
responses (upward and downward deflections superimposed on the holding
current), the amplitudes of which are directly proportional to the
membrane conductance. Application of 10 nM GLP-1(7-37)
for 10 s induced an inward shift of the holding current (I
)
and a large increase of membrane conductance (trace 1), the
time course of which matched the rise of
[Ca
] (trace 2). Panel B,
perforated patch measurements of membrane current obtained from a rat
-cell equilibrated in 7.5 mM glucose and held under
voltage clamp at -70 mV. Application of 10 nM GLP-1(7-36)amide for 10 s produced an inward shift of the
holding current (I
). Deflections superimposed on the inward
current are membrane current transients evoked by shifting (1 mV/ms)
the membrane potential from -70 to 0 mV. This ramp stimulus
protocol was used to evaluate the current as a function of voltage (I
- V) relationship for I
(inset; see
``Results''). Not illustrated are control current transients
evoked prior to application of GLP-1. I
exhibited a
reversal potential of -26 mV (inset).
resulted from the activation of
ion channels, as demonstrated by measurements of membrane conductance.
Changes in conductance associated with the appearance of I
were monitored by determining the amplitude of steady-state
inward (I
) and outward (I
) currents
generated by ±10-mV shifts in membrane potential of 1-s duration
evoked from a holding potential of -70 mV. A large conductance
increase in response to GLP-1 was observed (Fig. 7A, trace 1), as indicated by the increased amplitude of the
evoked inward and outward currents). The time course of the conductance
change matched that of the inward current and the associated rise in
[Ca
]
, suggesting a
causal relationship between these phenomena.
-cell by using a ramp stimulus protocol to obtain a
current-voltage relationship (Fig. 7B). Cells were
bathed in an extracellular solution containing 5 mM tetraethylammonium, 1 µM tetrodotoxin, and 1
µM nifedipine to block voltage-dependent
K
, Na
, and Ca
channels. Voltage ramps of 70-ms duration from -70 to 0 mV
were applied prior to and during the application of GLP-1. The membrane
current generated before stimulation with GLP-1 was subtracted from the
current evoked during exposure to the peptide, and the difference
current was plotted as a function of membrane potential (inset of Fig. 7B). The resultant current-voltage
relationship indicated an apparent reversal potential of -26 mV.
The mean reversal potential based on four such experiments was
-26 ± 4 mV (n = 4 HIT-T15 cells).
]
even though the
membrane potential was maintained at -70 mV (data not shown).
These actions of 8-Br-cAMP were also observed in rat
-cells (n = 5) and
TC6 cells (n = 10, data not
shown). Furthermore, the reversal potential for the 8-Br-cAMP-induced
inward current (-28 ± 2.3 mV, n = 4)
measured in HIT-T15 cells approximated that of GLP-1, as expected if
both agents activate the same type(s) of ion channels.
represents a
La
-blockable Na
current, the
activation of which is sensitive to intracellular Ca
. Panel A, the inward current and increased membrane conductance
observed in response to 1 mM 8-Br-cAMP applied to an HIT-T15
cell for 10 s was blocked by focal application of
Na
-free extracellular solution (NMG) or by bath
application of 100 µM La
. The membrane
was voltage clamped at -70 mV in the perforated patch
configuration, and the membrane conductance was monitored by applying
±10-mV voltage steps. Panel B, intracellular
administration of Ca
chelators to HIT-T15 cells
blocked the inward current response to 1 mM 8-Br-cAMP. Prior
to initiating perforated patch recordings, cells were incubated for 1 h
in 20 µM BAPTA/AM. For whole cell recordings, the cells
were dialyzed with an intracellular solution containing 5 mM EGTA (Ca
-free) or 160 nM Ca
. Each bar of the histogram
represents measurements obtained from five cells. Error bars indicate mean ± S.D. The membrane was voltage clamped at
-70 mV. In panels A and B the extracellular
solution contained 0.8 mM glucose.
I
The inward current observed in response to
GLP-1 could result from activation of two different signaling
mechanisms. Such a current might be secondary to the GLP-1-induced rise
of [Ca May Result from Activation of
Nonselective Cation Channels by cAMP and
Ca
]
since, in
insulinoma cells, Ca
is reported to activate
nonselective cation channels (Ca-NS channels; terminology of (59) ), the properties of which conform in some ways to the
findings presented here. Alternatively, GLP-1 might exert a more
immediate stimulatory effect, possibly involving the direct activation
of nonselective cation channels by cAMP, as has also been reported for
insulinoma cells(43) . Both concepts are consistent with three
additional sets of observations obtained in HIT-T15 cells.
was inhibited by exposure of cells to an extracellular
solution containing either NMG (an impermeant cation substituted for
Na
) or the cation channel blocker La
(Fig. 8A). Second, activation of I
was blocked by prior incubation of the cells for 1 h in a 20
µM concentration of the membrane-permeant Ca
chelator BAPTA/AM (n = 5), which will buffer
cytosolic Ca
, thereby blocking activation of
nonselective cation channels by a rise of intracellular Ca
(Fig. 8B). Third, activation of I
was not observed when cells were dialyzed in the standard whole
cell configuration (no nystatin; ruptured membrane patch) using a
pipette solution that was Ca
-free and which contained
5 mM EGTA (Fig. 8B). In contrast, I
was observed when the cells were dialyzed with a pipette solution
containing 160 nM Ca
(Fig. 8B). These findings suggest that I
represents a La
-blockable Na
current, the appearance of which results from the opening of
nonselective cation channels activated not only by cAMP, but also by
intracellular Ca
.
-cell glucose signaling system, acting to regulate enzymes and ion
channels that normally mediate the stimulatory actions of glucose on
insulin synthesis and secretion(26, 27) . Here we
demonstrate that a novel Ca
signaling pathway is
likely to mediate some of these actions of GLP-1 and that this pathway
may serve as an important effector mechanism at which signal
transduction cross-talk occurs between the cAMP and glucose signaling
systems.
]
in
-cells
exhibits several unusual features. The response to GLP-1 is augmented
by D-glucose, is dependent on extracellular Na
and Ca
, and is mimicked by forskolin, IBMX, and
cAMP analogs. Moreover, the GLP-1-induced rise of
[Ca
]
is associated
with, but is not dependent on, membrane depolarization and appears to
be the result of I
. I
is associated with
an increased membrane conductance and represents, at least in part, a
Na
current that is blocked by extracellular
La
or by intracellular application of the
Ca
chelators EGTA or BAPTA/AM.
proteins (60) and stimulates adenylyl cyclase, thereby
accelerating conversion of ATP to cAMP. We propose that this catalytic
process is dependent on extracellular Na
and that the
subsequent binding of cAMP to cyclic nucleotide-regulated nonselective
cation channels (or a protein closely associated with the channel)
results in channel activation, thereby generating I
.
Activation of these channels by cAMP is also proposed to require
intracellular Ca
. The rise of
[Ca
]
which accompanies
I
is achieved by stimulation of at least two distinct
Ca
signaling pathways. First, the membrane
depolarization that is a direct consequence of I
results
in activation of VDCCs, thereby raising
[Ca
]
. Second, a rise
of [Ca
]
is observed
even under conditions in which the membrane potential is
voltage-clamped at values (-100 to -70 mV) negative to the
activation threshold of VDCCs. Although the nature of this additional
rise of [Ca
]
remains
to be determined, it may signify the mobilization of Ca
from intracellular stores, as well as Ca
influx
via nonselective cation channels and/or membrane transporters (see
below). Acting in concert, these Ca
signaling
pathways are proposed to contribute to the stimulatory actions of GLP-1
on insulin secretion from
-cells.
] in
-cells. Receptor occupancy by GLP-1 leads to activation of
G
proteins and stimulation of adenylyl cyclase. This
step results in cAMP production and requires extracellular
Na
. The cAMP activates Ca-NS channels that are also
activated by cytosolic Ca
and which fail to respond
to cAMP when [Ca
] is very low. The opening
of these channels generates I
, an inward Na
current. I
results in membrane depolarization
(
V) and raises [Ca
] by
activation of VDCCs. I
may also promote a rise of
[Na
] which slows or reverses
Na
/Ca
exchange. The rise of
[Ca
] which is secondary to the slowing of
Na
/Ca
exchange may then, in turn,
promote Ca
-induced Ca
release (CICR) from intracellular Ca
stores.
Ca
-induced Ca
release may also
result from the initial rise of [Ca
] due to
the opening of VDCCs. Release of Ca
from
intracellular stores may also be favored by a more direct effect of
cAMP on them.
. As summarized above, omission of extracellular
Na
blocks the ability of GLP-1 to stimulate cAMP
production even though the binding of GLP-1 to its receptor remains
unaffected. This observation is reminiscent of a previous report that
Na
(5-100 mM) enhances
-adrenergic
receptor-mediated stimulation of adenylyl cyclase(61) .
Furthermore, the stimulatory action of forskolin on adenylyl cyclase is
also facilitated by Na
(61) . Therefore,
Na
can act as a positive regulator of the
receptor-G
-cyclase complex, as was reported previously for
receptor-G
-cyclase interactions(62, 63) .
Na
enhances receptor-mediated stimulation of GTPase
activity(63, 64) , and it amplifies receptor-mediated
inhibition of adenylyl
cyclase(65, 66, 67, 68) . A similar
regulatory action of Na
on
receptor-G
-cyclase coupling in
-cells might then
explain why GLP-1 fails to stimulate cAMP production in the absence of
Na
.
blocks the actions of GLP-1, forskolin, and
8-Br-cAMP to activate I
and to raise
[Ca
]
. Such
observations are not unique to single, isolated
-cells since
Fridolf and Ahren reported that GLP-1 increased
[Ca
]
and stimulated
insulin secretion from rat islets in a Na
-dependent
manner(32, 42) , actions that clearly resemble the
effects of GLP-1 reported here. However, since these previous studies
did not include an electrophysiological analysis, no correlation was
made between the GLP-1-induced rise of
[Ca
]
and the effects
of GLP-1 on membrane potential or current. Based on our own findings,
the most likely explanation for these observations is that
Na
is required not only for stimulation of cAMP
production by GLP-1 but also for generation of I
, which
represents, at least in part, a Na
current.
remains to be
determined. The rise of [Ca
]
which accompanies I
is observed not only in
response to the protein kinase A agonists 8-Br-cAMP and Sp-cAMP-S, but
also in response to Rp-cAMP-S, a protein kinase A antagonist.
Therefore, it appears likely that this response is initiated by the
binding of cAMP to a target (possibly the channel itself) other than
protein kinase A. Precedent for such a conclusion exists given that the
effects of cAMP on cyclic nucleotide-regulated Ca-NS channels in
excised membrane patches of CRI-G1 insulinoma cells are reportedly
independent of protein kinase A(43) . Furthermore, we find that
activation of I
by 8-Br-cAMP is observed even under
conditions in which diffusional exchange occurs between the patch
pipette (containing 160 nM Ca
) and the
cytosol, a configuration that disrupts protein kinase A-mediated
signaling by favoring ``wash out'' of enzymes and cofactors.
and K
are known to be expressed in the CRI-G1 insulinoma cell
line(43) . To date, the existence of such channels has yet to
be explored in
-cells or other insulinoma cell types. In rat
-cells and HIT-T15 cells we find that I
exhibits a
reversal potential of -26 mV, a value indicative of a permeation
pathway that does not strongly differentiate between Na
and K
, as is the case for Ca-NS channels. The
Ca-NS channels expressed in CRI-G1 cells are also noteworthy in that
they are stimulated by intracellular Ca
(59) .
This may also be the case for HIT-T15 cells since intracellular
application of the Ca
chelators EGTA or BAPTA/AM
blocks the activation of I
by 8-Br-cAMP. In marked
contrast, the response to 8-Br-cAMP is supported by intracellular
application of a solution containing 160 nM free
Ca
. Evidently, there is a requirement for some
minimal level of cytosolic Ca
for activation of
I
to occur. These findings are as expected if I
represents an inward current carried by Na
through cation channels activated not only by cAMP but also by
intracellular Ca
.
results in membrane depolarization and an increase of
[Ca
]
due, in part, to
activation of VDCCs (Fig. 9). On the basis of pharmacological
criteria, it was suggested previously that L-type VDCCs play a dominant
role in determining the magnitudes of the
[Ca
]
responses to
GLP-1 and 8-Br-cAMP in the
-cell
system(30, 31, 37) . Surprisingly, we find
that the actions of GLP-1 and 8-Br-cAMP on
[Ca
]
are only
partially diminished by the L-type Ca
channel
antagonists nifedipine, nimodipine, and verapamil. Moreover, the
ability of GLP-1 and 8-Br-cAMP to raise
[Ca
]
is reduced but
not blocked by voltage clamping the membrane to potentials negative to
the activation threshold of VDCCs. These findings are clear indications
that the effects of GLP-1 and cAMP analogs on
[Ca
]
are mediated not
only by VDCCs, but also by an as yet to be fully characterized
voltage-independent process that plays a major role in determining the
magnitude of the [Ca
]
response.
as well as
by monovalent cations(69) . Therefore, under conditions of
hyperpolarizing voltage clamp, Ca
entry might
contribute to the generation of I
and the ensuing rise
of [Ca
]
. Consistent
with this concept, we find that GLP-1 and 8-Br-cAMP are without effect
on [Ca
]
when the
extracellular solution does not contain Ca
. However,
8-Br-cAMP also fails to raise
[Ca
]
when the
extracellular solution contains Ca
but not
Na
. It is conceivable that Na
serves
to support permeation of such channels by Ca
so that
under Na
-free conditions, no Ca
current is generated. If, however, Ca
entry
does not contribute to I
, the question then
arises as to how GLP-1 or 8-Br-cAMP raises
[Ca
]
under conditions
in which the cells are voltage-clamped at membrane potentials negative
to the activation range of VDCCs. One possibility is that
I
, a Na
current, produces a rise of
[Na
]
, which then
indirectly raises [Ca
]
by slowing or reversing the process of plasma membrane
Na
/Ca
exchange (Fig. 9).
]
by stimulating
the release of Ca
from intracellular stores. Such a
process might be blocked by transient exposure to a
Ca
-free extracellular solution (as, for example, Fig. 4A) which disrupts Ca
-dependent
mobilization of intracellular Ca
stores by cAMP.
Furthermore, the release of Ca
from such stores might
also be disrupted by removal of extracellular Na
(as,
for example, Fig. 3B). Under Na
-free
conditions, activation of I
will not slow
Na
/Ca
exchange since no increase of
[Na
]
is likely.
Therefore, the increase of [Ca
]
which results from a slowing of the exchanger will not
occur, and the process of Ca
-induced Ca
release will not be initiated (Fig. 9). Whatever the exact
mechanism, it appears that initiation of the rise of
[Ca
]
by GLP-1 does not
require Ca
release from ryanodine-sensitive
intracellular Ca
stores since we find that
pretreatment of cells with ryanodine fails to block the response.
]
through
activation of a signaling system not involving effects on
I
ATP has at least one important ramification. GLP-1
augments insulin secretion in non-insulin-dependent diabetics, even
under conditions in which the sulfonylurea drugs such as glyburide
(which inhibits I
ATP) fail to stimulate insulin secretion
(sulfonylurea failure)(18, 21) . This observation
suggests that one therapeutic advantage of GLP-1 relative to that of
sulfonylureas in the treatment of non-insulin-dependent diabetes is
that GLP-1 triggers a rise of
[Ca
]
, insulin
secretion, and a lowering of blood glucose, even under conditions in
which sulfonylurea receptors and ATP-sensitive potassium channels no
longer play a dominant role in the regulation of
-cell
stimulus-secretion coupling. Therefore, activation of I
by GLP-1 may serve as a reserve mechanism of action, one that
complements its previously reported inhibitory effects on
I
ATP(26) . This would then explain why the
glucagon-like peptides retain their biological activity and augment
insulin secretion even under conditions in which sulfonylureas are no
longer effective.
ATP, ATP-sensitive potassium
current; VDCC(s), voltage-dependent Ca
channel(s);
[Ca
], free intracellular
Ca
; I
, inward current activated by
cAMP and GLP-1; NMG, N-methyl-D-glucamine; PACAP-27,
pituitary adenylyl cyclase-activating peptide-27; Sp- and Rp-cAMP-S,
Sp- or Rp-adenosine 3`,5`-cyclic monophosphothionate triethylamine;
IBMX, isobutylmethylxanthine; 8-Br-cAMP, 8-bromo-cAMP; I
,
holding current; Ca-NS, Ca
-activated nonselective
cation channel; BAPTA/AM, 1,2-bis(2-aminophenoxy)ethane N, N, N`, N`-tetraacetic acid/acetoxymethyl.
We thank Dr. Bernard Thorens for the receptor cDNA,
Dr. Shimon Efrat for the TC6 cells, Dr. Douglas Tillotson of
IonOptix Corp. for excellent technical support, Maurice Castonguay for
preparation of cell cultures, and Heather Herman for assistance with
the cAMP assays.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.