From the Diabetes Unit and the ¶ Laboratory of
Molecular Endocrinology,
Howard Hughes Medical Institute,
Massachusetts General Hospital, Harvard Medical School,
Boston, Massachusetts 02114
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ABSTRACT |
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Glucagon-like peptide-1 (GLP-1) is an
intestinally derived insulinotropic hormone currently under
investigation for use as a novel therapeutic agent in the treatment of
type 2 diabetes mellitus. In vitro studies of pancreatic
islets of Langerhans demonstrated that GLP-1 interacts with specific
Glucagon-like peptide-1
(GLP-1)1 is a potent blood
glucose-lowering hormone that stimulates secretion of insulin from
pancreatic The GLP-1 receptor located on We have focused on early events in GLP-1 signal transduction in order
to ascertain how the rise of
[Ca2+]i that serves as a trigger
for secretion of insulin is achieved. In general, the alterations of
[Ca2+]i observed upon exposure of
More poorly understood is the fast transient increase of
[Ca2+]i observed in response to
GLP-1 (18, 19). Here we report that this action of the hormone requires
the presence of extracellular Ca2+, can occur independent
of any change of membrane potential, and is reproduced by
pharmacological activators of cAMP signaling. Typically, such transient
effects of hormones on [Ca2+]i are
considered to be a consequence of the mobilization of Ca2+
stores. GLP-1 might stimulate release of inositol trisphosphate (IP3)-sensitive Ca2+ stores because it
increases inositol phosphate production in COS-7 cells transfected with
the recombinant GLP-1 receptor (26) or in Xenopus oocytes
injected with GLP-1 receptor mRNA (27). However, GLP-1 has only a
small effect on inositol phosphate production in freshly isolated
islets (24, 28) or HIT-T15 insulinoma cells (16). Therefore, an issue
of central importance to our understanding of GLP-1 signal transduction
concerns exactly how cAMP interacts with It has been proposed that Ca2+ is mobilized by GLP-1 during
the process of Ca2+-induced Ca2+ release
(CICR), whereby influx of Ca2+ across the plasma membrane
triggers release of Ca2+ from Ca2+ stores
regulated by ryanodine receptor Ca2+ release channels (20).
This hypothesis is controversial due to conflicting findings concerning
what role ryanodine receptors play as regulators of Here we report functional expression of ryanodine receptors in
Preparation of Human, Rat, and Mouse Islet Cell
Cultures--
Human islets were obtained from Dr. C. Ricordi (Diabetes
Research Institute, University of Miami School of Medicine, Miami, FL).
Rat islets were isolated from pentobarbital-anesthetized male
Sprague-Dawley rats (200-250 g; Charles River Laboratories, Wilmington, MA) according Lacy and Kostianovsky (41) by digestion with
collagenase (Roche Molecular Biochemicals). Mouse islets were isolated
by digestion of pancreata obtained from male C57BL/6J mice (Jackson
Laboratories, Bar Harbor, ME). Islets were dispersed into single cell
suspensions by digestion with trypsin-EDTA, and by trituration through
a fire-polished Pasteur pipette. Cell suspensions were plated onto
glass coverslips (25CIR-1; Fisher) coated with 1 mg/ml concanavalin A
(type V; Sigma), which facilitates adherence of Preparation of Insulinoma Cell Cultures--
BTC3 and BTC6 cells
(passages 62-78 and 24-34, respectively) were obtained from Dr. S. Efrat (Albert Einstein College of Medicine, New York, NY). MIN6 cells
(passages 25-35) were provided by J. Miyazaki (Kyoto University,
Japan). HIT-T15 cells (passages 65-70) were obtained from the American
Type Culture Collection (Rockville, MD). Cell cultures were maintained
on plastic tissue culture dishes (Falcon 3003; Becton Dickinson,
Franklin Lakes, NJ) containing media supplemented with 2 mM
glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. BTC3
and BTC6 cells were maintained in Dulbecco's modified Eagle's medium
containing 25 mM glucose, 15% heat-inactivated horse
serum, and 2.5% FBS. MIN6 cells were maintained in Dulbecco's
modified Eagle's medium composed of 25 mM glucose, 15%
FBS, and 74 µM 2-mercaptoethanol. HIT-T15 cells were
maintained in Ham's F-12K medium containing 10 mM glucose, 10% horse serum, and 2.5% FBS.
RT-PCR Analyses of Ryanodine Receptor Isoforms--
Total RNA
was extracted from rat islets and insulinoma cell cultures using
guanidinium thiocyanate. The RNA was fractionated by density gradient
centrifugation on a CsCl cushion, and poly(A) mRNA was isolated by
chromatographic separation on an oligo(dT) affinity column. First
strand cDNA synthesis was initiated using oligo(dT) primers and
avian myeloblastosis virus reverse transcriptase (Copy Kit; Invitrogen
Corp., Carlsbad, CA). A cDNA synthesis reaction to which no reverse
transcriptase was added served as a negative control. PCR primer pairs
corresponding to RYR isoforms were identified by analysis of the
published sequences (42, 43) of RYR cDNAs using Oligo version 4.0 primer analysis software (National Biosciences Inc., Plymouth, MN). The
design of the PCR primers is listed in Table I. PCR reactions were
catalyzed using recombinant Taq DNA polymerase, PCR reaction
buffer, and dNTPs supplied by TaKaRa Biologicals (Shiga, Japan). The
thermal cycling parameters consisted of an initial denaturation step
for 5 min at 95 °C, followed by 36 cycles consisting of: 30 s
at 95 °C, 60 s at 55 °C, and 90 s at 72 °C. The
final extension step was for 5 min at 72 °C. PCR products were
resolved by 1% agarose gel electrophoresis, and DNA was stained with
ethidium bromide for fluorescence detection. PCR products corresponding
to RYR were extracted from agarose gels by solubilization in NaI and by
extraction with silica (GeneClean II; Bio 101 Inc., La Jolla, CA). The
purified PCR products were ligated into the pCRII cloning vector by the
T/A cloning method (Invitrogen Corp.). Ligation products were used to
transform INV Fluorescence Microscopy, Immunocytochemistry, and FluorImager
Analyses--
The binding of BODIPY FL-X ryanodine (Molecular Probes
Inc., Eugene, OR) to its receptor was evaluated by fluorescence
microscopy using primary cultures of dispersed islet cells. An Eclipse
E600 upright microscope (Nikon, Melville, NY) equipped with 20, 40, and
100× Plan Fluor objectives was interfaced with a digital video imaging
system comprising a Photometrics Ltd. Sensys cooled CCD camera (Tucson,
AZ) and a MacIntosh G3 computer (Apple Computers, Cupertino, CA)
running IP Lab Spectrum software (Scanalytics Inc., Fairfax, VA). A
mercury lamp served as the excitation light source, and the emitted
light was filtered using standard fluorescein and rhodamine filter
sets. Individual islet cells were allowed to adhere to the numbered
grid surfaces of CELLocate coverslips (55-µm square size; Eppendorf,
Hamburg, Germany). The cells were fixed in 4% paraformaldehyde and
permeabilized using 0.1% Triton X-100. The fixed and permeabilized
cells were incubated at room temperature in Hanks' balanced salt
solution containing the specified concentration of BODIPY FL-X
ryanodine. Immunocytochemistry for detection of insulin-like
immunoreactivity was performed using 5% normal goat antiserum as a
blocking agent. The primary antiserum was guinea pig anti-insulin
(Dako, Carpinteria, CA) used at a dilution factor of 1:100. The
secondary antiserum was rhodamine-conjugated goat anti-guinea pig
(Jackson ImmunoResearch Laboratories, West Grove, PA). Quantitative
analyses of islet cell suspensions were performed using a Molecular
Dynamics FluorImager running ImageQuant software (Sunnyvale, CA). An
argon laser served as the excitation light source, and fluorescence was
imaged on 96-well tissue culture plates (Falcon 3072; ~20,000 islet
cells/well) using a fluorescein 530-nm discriminating filter set.
Estimates of RYR2 affinity were based on the use of permeabilized cells
where access of BODIPY FL-X ryanodine to its binding site was
unimpeded. Although the apparent Kd for fluorophore
binding was ~4 nM, higher concentrations of ryanodine
were required to block CICR in living cells, where access of ryanodine
to its binding site was impeded.
Measurement of Intracellular Ca2+
Concentration--
Cells were maintained on glass coverslips 24-48 h
prior to each experiment. The cell culture media was replaced with a
standard extracellular salt solution (SES) containing (in
mM): 138 NaCl, 5.6 KCl, 2.6 CaCl2, 1.2 mM MgCl2, 10 mM HEPES (pH 7.4), and
7.5 mM D-glucose. The SES was supplemented with
1 µM fura-2 acetoxymethyl ester (fura-2-AM; Molecular
Probes Inc.), 2% fetal bovine serum, and 0.02% Pluronic F-127 (w/v;
Molecular Probes Inc.). Cells were exposed to the fura-2 AM-containing
solution for 15 min at 22 °C. The loading solution was then removed,
and the cells were equilibrated in fresh SES for 10 min at 22 °C.
Experiments were performed at 32 °C using a Zeiss IM35 inverted
microscope (Thornwood, NJ) outfitted with a temperature-controlled
stage, a superfusion system, and a 100× Nikon UVF objective. Dual
wavelength excitation microspectrofluorimetry was performed
ratiometrically at 1-s intervals using a digital video imaging system
(IonOptix Corp., Milton, MA). Calibration of the raw fluorescence
values was performed as described previously (22) using fura-2
pentapotassium salt dissolved in calibration buffer solutions from
Molecular Probes (Calcium Calibration Kit 1 with Magnesium).
Insulin-containing Patch Clamp Electrophysiological Techniques--
The resting
potential and holding current were measured under current clamp or
voltage clamp using the tight seal, whole cell, perforated patch
configuration (13, 14). Pipettes were pulled from borosilicate glass
(Kimax-51; Kimble Glass Inc., Vineland, NJ), fire-polished (final tip
resistances of 2-3 megohms), and tip-dipped in a standard patch
pipette solution containing (in mM): 95 Cs2SO4, 7 MgCl2, and 5 HEPES (pH
7.4). Pipettes were then back-filled with patch pipette solution
supplemented with 240 µg/ml nystatin. The pipette solutions did not
contain fura-2 and were considered to be nominally
Ca2+-free. Patch pipettes were connected to the head stage
of an EPC-9 patch clamp amplifier (Heka Electroniks, Lambrecht,
Germany) interfaced with a personal computer running Pulse version 8.0 acquisition software (Instrutech Corp., Mineola, NY). The series
resistance (Rs) and cell capacitance
(Cm) were monitored following seal formation, and experiments were conducted when Rs declined
to 12-25 megohms and Cm increased to 10-40
picofarads. In voltage clamp experiments, Rs was
compensated for by 60%. Electrical access to the cytosol was confirmed
by noting approximately Preparation of Test Solutions--
GLP-1-(7-37), exendin-4, and
exendin-(9-39) were from Peninsula Laboratories (Belmont, CA).
8-Br-cAMP, nimodipine, ryanodine, caffeine, and thapsigargin were from
Sigma. (Rp)-cAMPS was from BioLog Life Sciences Institute
(Bremen, Germany). Test solutions were dissolved in SES prior to each
experiment. For studies examining the effects of peptides, the SES also
contained 0.05% human serum albumin (fraction V; Sigma) to protect
against their absorption to glass or plastic surfaces. Solutions to
which no Ca2+ was added were prepared by substituting
MgCl2 for CaCl2. Solutions containing 44 mM KCl were prepared by substituting KCl for NaCl on an
isosmotic basis. Test solutions were applied to individual cells from
micropipettes using a PicoSpritzer II pneumatic pressure ejection
system (General Valve Corp., NJ) as described previously (13).
BODIPY FL-X Ryanodine as a Fluorescent Probe for Detection of
Ryanodine Receptors--
We tested for expression of high affinity
ryanodine receptors in pancreatic
Specific binding of BODIPY FL-X ryanodine to human islet cells was
observed using a low concentration (1 nM) of the
fluorophore (Fig. 1, panel
A). A perinuclear pattern of fluorescence was observed (panel A), consistent with expression of
ryanodine receptors in a subcellular compartment that is likely to
include the endoplasmic reticulum. The affinity of BODIPY FL-X
ryanodine for these receptor sites appeared to be cell type-specific
because not all cells in a cluster of human islet cells were labeled
(arrows, panel B). BODIPY FL-X,
itself, was not effective in this assay (data not shown), thereby
confirming that the unconjugated fluorophore has no binding affinity.
These human islet cells labeled by BODIPY FL-X ryanodine included
authentic RT-PCR Analyses of Ryanodine Receptor Isoforms--
RT-PCR
analyses of the three known isoforms of RYR were performed using PCR
primers that match the published partial sequences (42, 43) of
cDNAs corresponding to mouse ryanodine receptors (Table
I). These primers target regions of RYR
cDNA that code for the M1-M4 membrane-spanning and carboxyl
terminus portions of the RYR protein (44). The samples of cDNA we
tested were derived from poly(A) mRNA extracted from rat islets.
Amplification of rat islet cDNA using an RYR2-specific primer set
(F04/R04) demonstrated that a product of the expected size (423 bp) was resolved by gel electrophoresis (Fig.
3A). In contrast, PCR products were not obtained when we amplified aliquots of a rat islet cDNA synthesis reaction mixture to which no reverse transcriptase was added
(Fig. 3A; negative control). The cloned 423-bp PCR product derived from rat islets was sequenced in both directions and evaluated by a National Center for Biotechnology BLAST analysis using the GenBank
data base. Determination of the nucleic acid sequence confirmed that it
was homologous (>90% identity) to the RYR2 sequence derived by
screening of a mouse cDNA library (42). This sequence corresponds
to sequence 13084-13506 (see Ref. 45) of rabbit cardiac RYR2.
It may be argued that our samples of rat islet cDNA are
contaminated by cDNAs derived from non-endocrine cell types that
express RYR2. Therefore, we sought to determine if RYR2 is detectable by RT-PCR of cDNA derived from insulin-secreting cell lines that serve as model systems for analysis of
We also sought to determine whether type 1 or type 3 isoforms of RYR
are detectable by RT-PCR in rat islets or insulinoma cells. Therefore,
RT-PCR was performed using RYR1- or RYR3-specific PCR primer sets
(Table I). As an internal control, the same batches of cDNA that
yielded RYR2 were used in each PCR reaction. Four primer sets
corresponding to RYR1 were tested. Of these, two sets (RYR1-F01/R01;
F02/R02) yielded faint PCR products of the appropriate sizes (298 and
385 bp, respectively) when amplifying cDNA from BTC3 cells but not
rat islets (data not shown). The 298-bp PCR product was sequenced,
whereby it was determined to be identical to that previously reported
for mouse RYR1 (42, 43). Also tested were four RYR3-specific primer
sets. Of these, only one set (RYR3-F01/R01) generated a PCR product of
the expected size (344 bp). This abundant PCR product was detected when
amplifying cDNA derived from rat islets, BTC3 cells, and mouse
brain (data not shown). Sequence analysis determined it not to
correspond to any known sequence in GenBank. Instead, it corresponded
most closely (93% identity) to mt19d02.rl, an expressed sequence tag (accession no. A178752) derived from a mouse cDNA library.
A Physiological Role for GLP-1 and Ryanodine Receptors in
These actions of GLP-1 resemble those effects known to be produced by
pharmacological activators of cAMP signaling. The membrane-permeant cAMP agonist 8-Br-cAMP produced a fast transient increase of
[Ca2+]i similar in time course and
magnitude to that produced by GLP-1 itself (Fig. 4A). A
similar effect was also obtained during exposure of
Detailed pharmacological studies were performed to characterize the
signaling system by which GLP-1 stimulates a fast transient increase of
[Ca2+]i. To achieve this goal, the
transient response was studied in isolation using cells for which no
sustained increase of [Ca2+]i was
observed. The action of GLP-1 was characterized with respect to: 1) the
requirement for a normoglycemic concentration of extracellular
D-glucose, 2) the ligand selectivity of the GLP-1 receptor,
3) the action of (Rp)-cAMPS as a membrane-permeant
antagonist of cAMP signaling, 4) the action of nimodipine as a
dihydropyridine antagonist of L-type Ca2+
channels, 5) the action of thapsigargin as a blocker of endoplasmic reticular Ca2+ uptake mediated by the SERCA class of
Ca2+ ATPases, and 6) the action of ryanodine as an
antagonist of CICR mediated by intracellular Ca2+ release channels.
We observed that equilibration of
It was also established that the stimulatory action of GLP-1 in this
assay exhibited pharmacological specificity characteristic of the
cloned GLP-1 receptor isolated from a rat islet cDNA library (3,
6). GLP-1-(7-37) and GLP-1-(7-36)amide isopeptides were effective,
whereas GLP-1-(8-37) was ineffective (Fig. 5B). Exendin-4, a peptide isolated from Heloderma, and which resembles in
structure GLP-1, also exhibited agonist activity (Fig. 5B).
In contrast, no effect of GLP-1-(7-37) was observed after treatment of
cells with exendin-(9-39) (Fig. 5B), a peptide antagonist
of the GLP-1 receptor.
A cAMP signaling system that regulates influx of Ca2+
through L-type Ca2+ channels is likely to be an
important determinant of this response. Equilibration of cells for 30 min at 37 °C in saline containing the cAMP antagonist
(Rp)-cAMPS blocked the fast transient increases of
[Ca2+]i measured in response to
GLP-1 or 8-Br-cAMP (Fig. 5C). The actions of GLP-1 and
8-Br-cAMP were also blocked by brief exposure of cells to a
Ca2+-free solution (Fig. 5C), or by treatment
with the L-type Ca2+ channel antagonist
nimodipine (Fig. 5C). It was also observed that this
signaling system targets an intracellular source of Ca2+
that is mobilized as a consequence of Ca2+ influx. No
effect of GLP-1 was observed after treatment of cells with the SERCA
Ca2+ ATPase inhibitor thapsigargin (Fig. 5C).
The stimulatory actions of GLP-1 and 8-Br-cAMP that resulted in a fast
transient increase of [Ca2+]i were
blocked by prior equilibration of islet cells for 5 min at 37 °C in
saline containing 44 mM KCl and 500 nM
ryanodine (Fig. 5D). This concentration of KCl produces
cellular depolarization and Ca2+ influx, thereby promoting
high affinity binding of ryanodine to Ca2+ release channels
that open as a consequence of CICR (44). No such antagonist action of
500 nM ryanodine was observed using islet cells not
pretreated with 44 mM KCl (Fig. 5D).
Pharmacological concentrations of ryanodine (20-100 µM)
were also observed to be effective provided that islet cells were
incubated for 30 min at 37 °C in saline containing 5.6 mM KCl (data not shown). Our use of 44 mM KCl
to "unmask" the potent antagonist action of ryanodine is
reminiscent of a previous study of RINm5F cells in which ryanodine was
reported to be an antagonist of Ca2+ signaling only under
conditions in which cells were pretreated with caffeine (48). Such
observations may help explain conflicting reports documenting the
effectiveness (48), limited effectiveness (49), or lack of effect (35,
50-53) of ryanodine as a blocker of Ca2+ signaling in
Caffeine as a Sensitizer of Ca2+-induced
Ca2+ Release Mediated by Ryanodine Receptors--
Caffeine
is a methylxanthine that acts as a co-agonist with cytosolic
Ca2+ to facilitate CICR mediated by ryanodine receptors in
cardiac myocytes (54). Whether ryanodine receptors also mediate
stimulatory effects of caffeine on
[Ca2+]i in
It is important to note that the action of caffeine was unrelated to
its previously reported inhibitory effect on K-ATP (51). Caffeine
produced an increase of [Ca2+]i in
HIT-T15 cells voltage clamped at The precise mechanism by which cAMP-elevating agents such as GLP-1
regulate Ca2+ signaling and insulin secretion in Evidence for Expression of Ryanodine Receptors in Pancreatic
Interpretation of findings obtained by RNase protection or RT-PCR
analysis is complicated by uncertainties concerning whether or not
there is functional expression of ryanodine receptor protein. Therefore, we tested for expression of ryanodine receptor binding sites
by fluorescence microscopy using the BODIPY FL-X ryanodine fluorophore.
Specific high affinity binding of BODIPY FL-X ryanodine was observed in
human islet, rat islet, and C57BL/6J mouse islet cells. The binding was
detected in permeabilized cells, was found to be saturable and
reversible, and was blocked by pretreatment of cells with
non-fluorescent ryanodine. Not all cells were labeled with BODIPY FL-X
ryanodine, but those that did included Caffeine as a Probe for Ca2+ Signaling Mediated by
Ryanodine Receptors--
It is well established that IP3
receptors play a highly significant role in the process by which
hormones and neurotransmitters mobilize Ca2+ stores in the
The findings of the present study contradict several of these
assertions and provide new evidence for an important role of ryanodine
receptors in An Interaction of cAMP, L-type VDCCs, and Ryanodine Receptors to
Regulate CICR--
We report that GLP-1 stimulates a fast transient
increase of [Ca2+]i under
conditions in which
These findings are interpreted in the model illustrated in Fig.
7, which shares important features with
previous models (20, 56, 69). GLP-1 stimulates production of cAMP and
activation of PKA, thereby exerting a dual effect. The hormone augments
influx of Ca2+ and an increase of
[Ca2+]i by promoting the opening
of L-type Ca2+ channels (step 1). It also
sensitizes RYR2 to stimulatory influences of cytosolic
Ca2+, thereby facilitating CICR from a Ca2+
store, the filling state of which is maintained by metabolism of
glucose (step 2). The fast transient increase of
[Ca2+]i that results is measured
in cells voltage clamped at
It should be emphasized that the model presented in Fig. 7 does not
rule out a role for IP3 receptors as mediators of GLP-1 signal transduction. GLP-1 has a small stimulatory effect on inositol phosphate production in rat islets (24, 28), and cAMP enhances Ca2+ release from IP3-sensitive stores in
hepatocytes (65, 66). Furthermore, permeation by Ca2+ of
the type 1 IP3 receptor is facilitated by PKA (67),
although such an effect has yet to be demonstrated for the type 3 IP3 receptor expressed in Conclusion--
It is now established that influx of
Ca2+ through L-type Ca2+ channels
produces an increase of [Ca2+]i
that serves as a powerful trigger for secretion of insulin. Exocytosis
occurs in localized patches of -cell G protein-coupled receptors, thereby facilitating insulin
exocytosis by raising intracellular levels of cAMP and
Ca2+. Here we report that the stimulatory influence of
GLP-1 on Ca2+ signaling results, in part, from
cAMP-dependent mobilization of ryanodine-sensitive
Ca2+ stores. Studies of human, rat, and mouse
-cells
demonstrate that the binding of a fluorescent derivative of ryanodine
(BODIPY FL-X ryanodine) to its receptors is specific, reversible, and of high affinity. Rat islets and BTC3 insulinoma cells are shown by
reverse transcriptase polymerase chain reaction analyses to express
mRNA corresponding to the type 2 isoform of ryanodine receptor-intracellular Ca2+ release channel (RYR2).
Single-cell measurements of
[Ca2+]i using primary cultures of
rat and human
-cells indicate that GLP-1 facilitates
Ca2+-induced Ca2+ release (CICR), whereby
mobilization of Ca2+ stores is triggered by influx of
Ca2+ through L-type Ca2+ channels.
In these cells, GLP-1 is shown to interact with metabolism of
D-glucose to produce a fast transient increase of
[Ca2+]i. This effect is
reproduced by 8-Br-cAMP, but is blocked by a GLP-1 receptor antagonist
(exendin-(9-39)), a cAMP antagonist ((Rp)-cAMPS), an
L-type Ca2+ channel antagonist (nimodipine), an
antagonist of the sarco(endo)plasmic reticulum Ca2+ ATPase
(thapsigargin), or by ryanodine. Characterization of the CICR mechanism
by voltage clamp analysis also demonstrates a stimulation of
Ca2+ release by caffeine. These findings provide new
support for a model of
-cell signal transduction whereby GLP-1
promotes CICR by sensitizing intracellular Ca2+ release
channels to the stimulatory influence of cytosolic
Ca2+.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cells, and which is under intensive clinical
investigation for use in treatment of type 2 diabetes mellitus (1, 2).
The two naturally occurring and biologically active isopeptides of GLP-1 with therapeutic potential are GLP-1-(7-37) and
GLP-1-(7-36)amide (3, 4). They are derived by post-translational
processing of proglucagon, and are synthesized and secreted by
enteroendocrine L-cells of the distal intestine. The timing
of the secretion of GLP-1 from these cells is such that circulating
levels of the hormone rise coincident with the postprandial increase of
blood glucose concentration. By binding to its receptors located on
-cells, GLP-1 synergizes with glucose to induce insulin secretion, thereby increasing the concentration of circulating insulin to a level
above that attributable to glucose alone. This augmentation of
glucose-stimulated insulin secretion by the gut hormone GLP-1 is termed
the incretin effect and plays a critical role in the maintenance of
systemic glucose homeostasis (5).
-cells is a G protein-coupled receptor
that mediates stimulatory effects of the hormone on adenylyl cyclase
(6). GLP-1 raises intracellular levels of cyclic cAMP, activates
protein kinase A (PKA), and exerts pleiotropic effects on
-cell
signal transduction (7-9). PKA-mediated protein phosphorylation
interacts with metabolism of D-glucose at a late step in
stimulus-secretion coupling to facilitate exocytosis of insulin-containing secretory granules (10-12). GLP-1 also produces cellular depolarization (13-15) and a large increase of
[Ca2+]i (14, 16-20), effects
reproduced by activators of cAMP signaling (21-23). The best evidence
available indicates that the GLP-1-induced rise of
[Ca2+]i serves as an important
trigger for exocytosis of insulin. The vital role Ca2+
plays in this process is emphasized by studies demonstrating that the
insulin secretagogic effect of GLP-1 is markedly attenuated by
treatments that prevent the rise of
[Ca2+]i (16, 24).
-cells to GLP-1 can be described as having transient (seconds) or
sustained (minutes) kinetics. The sustained rise of
[Ca2+]i results from influx of
Ca2+ that is a consequence of three distinct mechanisms.
First, GLP-1 potentiates glucose-induced closure of ATP-sensitive
K+ channels (K-ATP) (13), thereby generating cellular
depolarization, activation of voltage-dependent
Ca2+ channels (VDCCs), and influx of Ca2+.
Second, GLP-1 exerts a direct stimulatory influence on the entry of
Ca2+ through dihydropyridine-sensitive (L-type)
VDCCs (9). Third, GLP-1 stimulates the opening of
Ca2+-activated nonselective cation channels that are
permeant to Ca2+ as well as to Na+ (14, 25).
These three processes act in concert to augment oscillatory electrical
activity, Ca2+ influx, and pulsatile secretion of insulin
characteristic of whole islets of Langerhans.
-cell Ca2+
stores to promote their mobilization.
-cell
Ca2+ signaling. One objection raised concerns the uncertain
effectiveness of cyclic ADP-ribose (cADPR) in this system. cADPR is a
candidate second messenger that mobilizes ryanodine-sensitive
Ca2+ stores in some cell types (29). It is derived from
-NAD+ via an enzymatic process catalyzed by an
ADP-ribosyl cyclase designated CD38 (30). Okamoto and co-workers
reported that an IP3-insensitive Ca2+ store was
mobilized by cADPR in rat islet microsomes (31-33). However, cADPR
failed to act in ob/ob mouse
-cells or RINm5F insulinoma
cells (34-38). Ca2+ signaling in rat
-cells was also
not influenced by an antagonist of cADPR (37, 39), and levels of cADPR
in rat islets were insensitive to alterations of glucose concentration
(40), findings that contradict the Okamoto hypothesis. Therefore, the
significance of ryanodine receptors as mediators of GLP-1 signal
transduction remained unclear.
-cells derived from human, rat, and C57BL/6J mouse islets. Assays of
islet mRNA by the reverse transcriptase-polymerase chain reaction
(RT-PCR) indicated a preferential expression of the type 2 isoform of
ryanodine receptor (RYR2) in these tissues. The subcellular location of
ryanodine receptors was visualized in
-cells by fluorescence microscopy using BODIPY FL-X ryanodine as a fluorophore.
Ca2+ imaging and patch clamp analyses of
-cells
demonstrated that GLP-1 produces a fast transient increase of
[Ca2+]i as a consequence of CICR
initiated by influx of Ca2+ through L-type
VDCCs. It is concluded that this process of CICR is mediated by a
cAMP-signaling system that targets a ryanodine-sensitive source of
intracellular Ca2+.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cells to glass
surfaces. Cell cultures were maintained in a humidified incubator (95%
air, 5% CO2) at 37 °C in RPMI 1640 culture media
containing 11.1 mM glucose, 2 mM glutamine,
10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 µg/ml streptomycin.
F' cells (Invitrogen Corp.), and transformants were
isolated by antibiotic resistance selection on agar plates containing
ampicillin. Individual clones were selected and amplified by
inoculation of Luria broth. Cloned plasmid DNAs were extracted from
bacterial cultures using the Rapid Pure Miniprep procedure (Bio 101 Inc.). Determination of plasmid DNA sequences containing RYR PCR
products was accomplished by the dideoxynucleotide sequencing method of
Sanger in combination with polyacrylamide gel electrophoresis
(Sequenase version 2.0 DNA sequencing kit; U. S. Biochemical
Corp.).
-cells were positively identified at the end of
each experiment by fluorescence microscopy in combination with immunocytochemistry.
60-mV resting potential, and by noting an
increase of [Ca2+]i in response to
a voltage step from
70 to 0 mV. Experiments were rejected if such a
rise of [Ca2+]i was not observed.
Measurements of membrane potential and current were digitized using an
Instrutech VR-10A digital data recorder and stored on magnetic tape for
subsequent analysis using pClamp version 6.0 software (Axon Instruments
Corp., Foster City, CA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cells by using the fluorophore
BODIPY FL-X ryanodine. Cells derived from dispersed human, rat, or
mouse islets were allowed to adhere to coverslips, on which was etched
a numbered grid system. This allowed positive identification of
individual cells by microscopic inspection. Cells were fixed with
paraformaldehyde, permeabilized with Triton X-100, and bathed for
1 h at 22 °C in saline containing BODIPY FL-X ryanodine. A
microscope-based digital imaging system was used to acquire images of
fluorescence attributable to binding of BODIPY FL-X ryanodine to its receptors.
-cells, as demonstrated by their co-expression of
insulin-like immunoreactivity (cf. panels C and D). Quantitative analyses of human islet
cells using a FluorImager demonstrated that the binding of BODIPY FL-X
ryanodine was saturable and reversible (Fig.
2A). Pretreatment of human
islet cells with 1 µM non-fluorescent ryanodine blocked
the subsequent binding of BODIPY FL-X ryanodine to its receptor (Fig.
2B). The IC50 for displacement of binding was
determined to be ~4 nM. These experiments conducted with
human islet cells were reproduced successfully using islets derived
from Sprague-Dawley rats and C57BL/6J mice (data not shown).
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Fig. 1.
Labeling of human pancreatic islet cells by
fluorescent BODIPY FL-X ryanodine. Primary cultures of human islet
cells were fixed with paraformaldehyde, permeabilized with Triton
X-100, and incubated at 22 °C for 1 h in Hanks' buffered
saline containing 1 nM BODIPY FL-X ryanodine. A pseudocolor
image of fluorescence due to binding of the fluorophore demonstrated a
perinuclear pattern of labeling (panel A; original
magnification, ×40). Imaging of a cluster of islet cells demonstrated
that not all cells were labeled (panel B, arrows;
original magnification, ×20). A double-labeling technique demonstrated
that a single human -cell labeled by BODIPY FL-X ryanodine
(panel D; original magnification, ×100) also contained
insulin-like immunoreactivity as visualized using a
rhodamine-conjugated secondary antiserum (panel C; original
magnification, ×100).
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Fig. 2.
The interaction of BODIPY FL-X ryanodine with
its receptor was saturable, reversible, and of high affinity.
Human islet cell suspensions maintained on 96-well tissue culture
plates were fixed, permeabilized, and incubated with saline containing
BODIPY FL-X ryanodine. Labeling of cells by the fluorophore was
quantitated using an argon laser-based imaging system. The maximal
intensity of fluorescence detected was assigned an arbitrary value of
1.0. Measurements were normalized to this value. A, cells
were exposed to 1 nM BODIPY FL-X ryanodine for 20, 40, 60, or 80 min at 22 °C. This led to a time-dependent
increase of labeling that decreased following removal of the
fluorophore from the extracellular solution (as indicated by
Wash). B, high affinity binding of BODIPY FL-X
ryanodine to its receptor was diminished by prior treatment of islet
cells for 60 min at 22 °C with the indicated concentration of
non-fluorescent ryanodine. A concentration of 4 nM
ryanodine reduced the binding of 1 nM BODIPY FL-X ryanodine
by 50%.
PCR primers used for amplification of RYR cDNAs
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Fig. 3.
RT-PCR analysis of RYR2 in rat islets and
mouse BetaTC3 insulinoma cells. A, RT-PCR amplification
of cDNA derived from rat islets or BetaTC3 cells was achieved using
RYR2-specific primer pair 4, designated as RYR2 F04/R04 (Table I). A
423-bp PCR product was resolved by gel electrophoresis, as indicated by
arrowheads in lanes designated as (+) Rev.
Trans. (for reverse transcriptase). No PCR product was detected
when amplifying a cDNA synthesis reaction mixture to which no
reverse transcriptase was added (lanes designated as ( ) Rev.
Trans.). B and C, RT-PCR amplification of
cDNA derived from rat islets (B) or BetaTC3 insulinoma
cells (C) was accomplished using RYR2-specific primer pairs
1 and 2, designated as RYR F01/R01 and RYR F02/R02 (Table I). The 303- and 258-bp PCR products were resolved by gel electrophoresis and are
indicated by arrowheads in lanes designated as
(+) Rev. Trans.
-cell function. To this end,
poly(A) mRNA was extracted from mouse BTC3 cells for use in
synthesis of cDNA. As was the case for rat islet cDNA, the RYR2-specific F04/R04 primer set generated a 423-bp PCR product when
using BTC3 cDNA as template (Fig. 3A). When cloned and
characterized, this BTC3 PCR product was found to be identical in
sequence to that reported for mouse RYR2. Three additional primer sets
were chosen to validate the presence of RYR2 mRNA in rat islets or insulinoma cells. RYR2 F01/R01 and F02/R02 primer pairs (Table I)
generated PCR products of the expected sizes (303 and 258 bp,
respectively) when used for amplification of cDNA from rat islets
(Fig. 3B) and BTC3 cells (Fig. 3C). Sequence
analysis confirmed that these PCR products correspond to RYR2. The 303- and 258-bp PCR products are homologous to sequences 14757-15059 and
14802-15059 of rabbit cardiac RYR2 (45). We also observed that the
RYR2 F03/R03 primer pair generated a product of the expected size (1179 bp) when amplifying cDNA from rat islets (data not shown). The RYR2
F03/R03 primers target a sequence in mouse and rabbit cardiac RYR2 that
encompasses the entire carboxyl terminus starting at position 14805 of rabbit RYR2 and extending into the 3'-untranslated region (42,
45).
-Cell
Ca2+ Signaling--
GLP-1 is proposed to influence
Ca2+ signaling in
-cells by mobilizing
ryanodine-sensitive Ca2+ stores (9, 20). To test this
possibility, measurements of [Ca2+]i were obtained from single
rat
-cells maintained in primary cell culture and equilibrated in
saline containing 7.5 mM D-glucose. Application
of 10 nM GLP-1-(7-37) produced a significant increase of
[Ca2+]i in 34 of 50 cells. These
responses were tabulated on the basis of kinetic parameters (Table
II). A monophasic response was measured
in 26 cells, whereas 8 cells exhibited a biphasic response. Monophasic
responses consisted of a fast transient increase of
[Ca2+]i lasting 20-30 s (Fig.
4A) or a slowly developing and sustained increase lasting several minutes (Fig. 4B).
Biphasic responses consisted of an initial transient increase of
[Ca2+]i followed by a sustained
effect (Fig. 4C).
Kinetic parameters of the GLP-1-induced rise of
[Ca2+]i
-cells to 10 nM
GLP-1-(7-37) under conditions in which cells were equilibrated in a
saline solution containing 7.5 mM D-glucose. Three types of
responses to GLP-1 were noted: transient, sustained, and biphasic.
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Fig. 4.
GLP-1-(7-37) and 8-Br-cAMP stimulated a
transient and/or sustained increase of
[Ca2+]i in primary cultures
of rat -cells. Ratiometric determinations
of [Ca2+]i were obtained by
microspectrofluorimetry of single
-cells loaded with fura-2.
A, a fast transient increase of
[Ca2+]i was observed during
repeated 30-s applications of 1 mM 8-Br-cAMP or 10 nM GLP-1-(7-37) (application of test solutions indicated
by horizontal bars). B, a slowly developing and
sustained increase of [Ca2+]i was
measured following a 30-s application of 10 nM
GLP-1-(7-37) (arrow indicates the time at which application
of GLP-1 commenced). C, a biphasic increase of
[Ca2+]i was observed following a
30 s application of 10 nM GLP-1-(7-37). D,
10 nM GLP-1-(7-37) produced a fast transient increase of
[Ca2+]i when applied to a
-cell
in which the membrane potential was held at
50 mV using the voltage
clamp technique. The action of GLP-1 was not associated with a change
of holding current (Ih, top
trace). Each panel indicates observations obtained from 4 different cells. The
-cell phenotype was established by detection of
insulin-like immunoreactivity (data not shown).
-cells to 10 µM forskolin or 10 µM of the cAMP agonist
(Sp)-cAMPS (data not shown). As expected for a signal transduction mechanism mediated by cell surface receptors, the action
of GLP-1 exhibited desensitization, whereas the effect of 8-Br-cAMP was
readily repeatable (Fig. 4A). Voltage clamp analysis using
the perforated patch technique demonstrated that a fast transient
increase of [Ca2+]i could also be
measured when GLP-1 was applied to cells in which the membrane
potential was maintained at
50 mV but not
80 mV (Fig.
4D). Under these conditions, the increase of
[Ca2+]i was not associated with a
detectable change of membrane current (Fig. 4D;
trace labeled Ih). Therefore, the
fast transient increase of [Ca2+]i
was unlikely to result from influx of Ca2+ as a consequence
of the activation of nonselective cation channels (14, 25) or cyclic
nucleotide-gated ion channels.
-cells in a normoglycemic
concentration (7.5 mM) of glucose allowed for a fast
transient increase of [Ca2+]i in
response to brief application (30 s) of GLP-1 or 8-Br-cAMP (Fig.
5A). Levels of
[Ca2+]i increased from 98 ± 30 nM to nearly 600 nM, an increase of 490 ± 65 nM for GLP-1 (Fig. 5A; Table II). In
marked contrast, the actions of GLP-1 and 8-Br-cAMP were diminished by
equilibration of cells in lower concentrations of glucose (Fig.
5A). Responses observed during exposure of cells to 2.0 or
0.8 mM glucose were measured not as a spike-like increase
of [Ca2+]i, but as a small
sustained effect (data not shown). This diminishment in response size
was accompanied by a decrease in the fraction of cells exhibiting any
response at all (Fig. 5A). Evidently, these fast transient
increases of [Ca2+]i are
all-or-none phenomena generated in a manner strictly dependent on
exposure of
-cells to a normoglycemic concentration of glucose. This
conclusion is consistent with the established role of glucose
metabolism as a positive regulator of Ca2+ uptake, thereby
maintaining the filling state of intracellular Ca2+ stores
(46, 47).
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Fig. 5.
Pharmacology of GLP-1 signal
transduction. Population studies in A-D refer to
actions of GLP-1 and 8-Br-cAMP to produce a fast transient increase of
[Ca2+]i in rat or human -cells.
Measurements are the mean ± S.D. increase of
[Ca2+]i above base line. Also
indicated is the fraction of cells that exhibited a
400
nM increase of
[Ca2+]i. Cultures were
equilibrated in the indicated concentrations of glucose for 30 min at
37 °C. GLP-1, exendin-4, exendin-(9-39), and 8-Br-cAMP were applied
to individual cells for 30 s. All other solutions were
administered by a superfusion system. Asterisks (*) indicate
a statistically significant difference from control (p
0.001, t test; control defined as the response to
GLP-1-(7-37) or 8-Br-cAMP when cells were bathed in saline containing
7.5 mM glucose alone). A, three sets of rat
islet cell cultures were equilibrated in 7.5, 2.0, or 0.8 mM glucose as indicated. Application of 10 nM
GLP-1-(7-37) or 1 mM 8-Br-cAMP (8-Br) produced a fast
transient increase of [Ca2+]i in
24% and 30% of cells equilibrated in 7.5 mM glucose,
respectively. Only a small increase of
[Ca2+]i was observed when the
saline contained 2.0 or 0.8 mM glucose. Under these
conditions the percentage of cells responding to either test substance
was reduced. B, GLP-1-(7-37), GLP-1-(7-36)amide, and
exendin-4 (10 nM each) were effective agonists, whereas
GLP-1-(8-37) was ineffective. The action of 10 nM
GLP-1-(7-37) was blocked by prior exposure of rat islet cells to 1 µM exendin-(9-39). C, application of 10 nM GLP-1-(7-37) or 1 mM 8-Br-cAMP failed to
increase [Ca2+]i when rat islet
cultures were pretreated for 30 min at 37 °C with 10 µM (Rp)-cAMPS (Rp-), 10 µM nimodipine (Nimod.), or 10 µM
thapsigargin (Thap.). 8-Br-cAMP was also ineffective when
cells were exposed for 120 s to a solution that contained no added
Ca2+ (0 Ca). D, application of 10 nM GLP-1-(7-37) or 1 mM 8-Br-cAMP to human
islet cells superfused with saline containing 5.6 mM KCl
failed to increase [Ca2+]i when
cultures were pretreated for 5 min at 37 °C with 44 mM
KCl and 500 nM ryanodine. No effect of ryanodine was
observed in cells not pretreated with 44 mM KCl.
-cells.
-cells remains
controversial. We examined what effect caffeine exerts on
Ca2+ signaling in HIT-T15 insulinoma cells, a hamster
-cell line that expresses GLP-1 receptors (16), L-type
VDCCs (16, 21), and the type 2 isoform of RYR (55). We found that
application of caffeine produced a fast transient increase of
[Ca2+]i in 25 of 30 cells tested
(Fig. 6A). This response was
similar in magnitude and time course to that produced by GLP-1 or
8-Br-cAMP in rat
-cells (cf. Figs. 4A and
6A). As might be expected for a process of CICR, the action
of caffeine was critically dependent on the initial
[Ca2+]i. There existed a positive
correlation between the [Ca2+]i
measured prior to and during application of caffeine (Fig.
6B). This correlation appears to explain our finding that the action of caffeine was potentiated by prior exposure of HIT-T15 cells to a depolarizing concentration (44 mM) of KCl that
produced a rise of [Ca2+]i on its
own (Fig. 6A).
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Fig. 6.
Sensitization of Ca2+-induced
Ca2+ release by caffeine. Caffeine (10 mM)
and KCl (44 mM) were applied to individual HIT-T15 cells
from micropipettes as indicated by bars. The extracellular
solutions contained 0.8 mM glucose. A,
potentiation of the caffeine-induced rise of
[Ca2+]i by prior stimulation with
KCl. An initial application of caffeine produced a small transient
increase of [Ca2+]i
(bar labeled 1) that diminished in size during
repeated administration of caffeine (bars 4,
7, and 10). Application of KCl produced a rise of
[Ca2+]i that was repeatable
(bars 2, 5, and 8). A
larger increase of [Ca2+]i was
observed when caffeine was applied immediately prior to recovery of the
response to KCl (bars 3, 6, and
9). This interaction of caffeine and KCl tended not to
exhibit tachyphylaxis. B, positive correlation of
[Ca2+]i measured prior to and
during application of caffeine. Elevated basal
[Ca2+]i favored a larger increase
of [Ca2+]i in response to caffeine
(n = 12 cells). C, voltage clamp analysis of
Ca2+-induced Ca2+ release. Caffeine produced a
transient increase of [Ca2+]i when
the membrane potential was maintained at 70 mV (bars
1 and 3). A gradual increase of
[Ca2+]i was observed when the
membrane potential was shifted from
70 to 0 mV for 100 ms at 2-s
intervals (arrow with dots). This
depolarization-induced rise of
[Ca2+]i allowed for a much larger
increase of [Ca2+]i in response to
caffeine (bar 2). D, antagonism by
ryanodine. The interaction of KCl and caffeine to produce an increase
of [Ca2+]i was demonstrated
(bars 1-3). Ryanodine (50 µM) was
then administered via a superfusion system and cells were equilibrated
at 32 °C for 15 min. Ryanodine blocked the interaction of KCl and
caffeine to produce a fast transient increase of
[Ca2+]i (bars
4-6). The trace for
[Ca2+]i was interrupted for ~300
s during which a light was turned on in order to monitor the
superfusion system.
70 mV (Fig. 6C). This
action of caffeine was potentiated by repetitive cellular depolarization to 0 mV (Fig. 6C). Such repetitive stimuli
generated influx of Ca2+ through VDCCs, thereby explaining
the gradual increase of [Ca2+]i
observed prior to application of caffeine (Fig. 6C; arrow with dots). From these observations, it is
evident that a close functional coupling existed between influx of
Ca2+ and the subsequent mobilization of caffeine-sensitive
Ca2+ stores in this cell type. Support for such a mechanism
of CICR is also provided by our finding that the interaction of 44 mM KCl and caffeine to produce a fast transient increase of
[Ca2+]i was attenuated by prior
exposure of HIT-T15 cells to ryanodine (Fig. 6D).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cells
continues to be a topic of considerable controversy. Here we present
findings in support of an important role for the type 2 isoform of
ryanodine receptor as a mediator of intracellular Ca2+
signaling activated by GLP-1-(7-37). RT-PCR analyses of rodent islet
cells and insulinoma cell lines indicated a preferential expression of
RYR2 in comparison to RYR1 or RYR3. These receptors were visualized by
fluorescence microscopy in fixed, permeabilized human, mouse, and rat
-cells using the BODIPY-FL-X ryanodine fluorophore. Functional
studies also demonstrated that application of ryanodine to
-cells
blocked the fast transient increase of [Ca2+]i observed in response to
GLP-1-(7-37). Taken together, these findings appear to substantiate an
important role for ryanodine receptors as mediators of GLP-1 signal transduction.
-Cells--
Islam and colleagues have used the RNase protection
assay to detect RYR2 mRNA in ob/ob mouse islets and BTC3
cells (56). The riboprobes used in that study were derived by in
vitro transcription of RYR cDNAs equivalent to partial
sequences of RYRs 1-3 from mouse (42, 43). These sequences were used
in the present study to identify RYR-specific PCR primers for RT-PCR
analyses of rat islets. Our findings complement those of Islam and
co-workers, whereby we demonstrate expression of RYR2 mRNA
corresponding to a region of the receptor that encompasses the
transmembrane spanning domains as well as the carboxyl terminus.
Takasawa and colleagues have also used RT-PCR to detect RYR2 mRNA
in islets obtained from C57BL/6J mice but not ob/ob mice or
RINm5F insulinoma cells (33). It was proposed that the ob/ob
model of diabetes/obesity is characterized by a defect of
Ca2+ signaling such that levels of RYR2 protein are reduced
(33). Should this prove to be the case, it may provide an explanation for the failure of previous studies to detect functional expression of
ryanodine receptors in ob/ob mouse islets (35, 37, 51-53) or RINm5F cells (Refs. 34, 36, and 50, but see Ref. 48).
-cells that stained
positively with anti-insulin antiserum. Therefore, BODIPY FL-X
ryanodine might serve as a useful fluorescent probe for future studies
examining the subcellular distribution of ryanodine receptors in
organellar structures of the
-cell.
-cell (57-60). It is also known that it is the type 3 isoform of
the IP3 receptor that is expressed at high levels in this
cell type (61). Much less well understood is the functional significance of IP3-insensitive Ca2+ stores. An
IP3-insensitive non-mitochondrial source of
Ca2+ was observed by Islam and co-workers to be mobilized
in a synergistic fashion during treatment of RINm5F cells with
thimerosal and caffeine (50). Although thimerosal and caffeine will
potentiate CICR from ryanodine-sensitive Ca2+ stores, the
mechanism by which caffeine exerts its action in RINm5F cells was
called into question. Studies of ob/ob mouse
-cells
failed to demonstrate a caffeine-sensitive source of intracellular Ca2+ (35, 51, 52). It was argued instead that caffeine
raised [Ca2+]i not by stimulatory
effects on ryanodine receptors, but by inhibitory actions on K-ATP,
whereby cellular depolarization produced influx of Ca2+ via
VDCCs (51).
-cell Ca2+ signaling. We observed GLP-1 and
caffeine to be effective under conditions of voltage clamp, producing a
fast transient increase of [Ca2+]i
in a manner independent of K-ATP (Figs. 4D and
6C). As expected for a process of CICR, the actions of
ryanodine and caffeine were shown to be facilitated by KCl-induced
depolarization (Figs. 5D and 6A) or by stepwise
changes of membrane potential (Fig. 6C). A positive
correlation was also established linking an increase of basal
[Ca2+]i to an increased
responsiveness of HIT-T15 cells to caffeine (Fig. 6B). Most
importantly, the actions of GLP-1 and caffeine were found to be blocked
by ryanodine (Figs. 5D and 6D). Such observations
provide compelling evidence for a ryanodine- and caffeine-sensitive
source of intracellular Ca2+ that is mobilized as a
consequence of CICR, a process activated in a direct manner
by GLP-1. Notably, these findings differ from those of Islam and
co-workers (56), who observed no such direct stimulatory action of cAMP
to release Ca2+ stores in mouse ob/ob
-cells.
-cells are equilibrated in a normoglycemic but
not hypoglycemic concentration of glucose. This action of the peptide
is mediated by receptors, the pharmacological properties of which match
those of the GLP-1 receptor cloned from rat islets. Analyses of the
signal transduction mechanism stimulated by GLP-1 demonstrates that its
effects on [Ca2+]i are mediated by
cAMP. The action of GLP-1 is blocked by the cAMP antagonist
(Rp)-cAMPS, and is mimicked by the agonist 8-Br-cAMP.
Evidence is also presented implicating two distinct types of
Ca2+ channels as targets of the GLP-1 signaling system.
Treatments that targeted L-type Ca2+ channels
(nimodipine) or Ca2+ release channels (ryanodine) render
GLP-1 ineffective. Finally, the source of Ca2+ mobilized by
GLP-1 is shown to be depleted by thapsigargin, thereby implicating the
SERCA class of Ca2+ ATPases in its regulation.
50 mV but not
80 mV becuse GLP-1
promotes partial activation of the L-type Ca2+
current, thereby allowing the opening of Ca2+ channels at a
membrane potential (
50 mV) slightly less negative than the resting
potential. This influx of Ca2+ is responsible for CICR
mediated by RYR2 sensitized to the stimulatory effects of cytosolic
Ca2+. Such a model is consistent with studies demonstrating
that PKA facilitates permeation by Ca2+ of
L-type Ca2+ channels (62) and ryanodine
receptors (63, 64).
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Fig. 7.
A model to explain stimulatory actions of
GLP-1 on -cell Ca2+
signaling. GLP-1 stimulates the production of cAMP and activation
of PKA. This effect is mimicked by 8-Br-cAMP and blocked by
(Rp)-cAMPS. PKA-mediated phosphorylation promotes the
opening of L-type voltage-dependent
Ca2+ channels and influx of Ca2+ (step 1). This
step is blocked by a Ca2+-free extracellular solution or by
treatment with nimodipine. PKA also sensitizes the type 2 isoform of
ryanodine receptor (RYR2) to stimulatory effects of cytosolic
Ca2+ (step 2). Steps 1 and 2 summate to trigger CICR. The
process of CICR is initiated by caffeine and is blocked by ryanodine,
thapsigargin, or an extracellular solution containing a low
concentration of glucose.
-cells. The model also does
not take into account actions of GLP-1 to induce a sustained increase
of [Ca2+]i, an effect upon which
the transient response may or may not be superimposed (cf.
Fig. 4, B and C). We propose that such sustained
actions of GLP-1 result from its inhibitory effect on ATP-sensitive
K+ channels (13) and/or its stimulatory action on
nonselective cation channels (14, 25).
-cell plasma membrane where secretory
granules co-localize with L-type Ca2+ channels
(68). Influx of Ca2+ through these channels triggers
exocytosis by producing a fast transient increase of
[Ca2+]i that is spatially
restricted (68). The efficacy of Ca2+ as a trigger for
exocytosis is limited by processes (Ca2+ buffering,
extrusion, re-uptake) that maintain
[Ca2+]i at a level subthreshold
for secretion. By facilitating Ca2+ influx and CICR, GLP-1
may allow for a localized and explosive increase of
[Ca2+]i that triggers secretion in
a highly efficient manner. Therefore, the functional significance of
CICR initiated by GLP-1 in the
-cell may be that it serves to
amplify any increase of [Ca2+]i
arising from entry of Ca2+ via the L-type
Ca2+ channels. In this manner, GLP-1 might stimulate two
interrelated and closely coupled processes (Ca2+ influx and
Ca2+ mobilization) that determine whether or not an
increase of [Ca2+]i is sufficient
to reach threshold for initiation of exocytosis.
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FOOTNOTES |
---|
* This work was supported by an American Diabetes Association research grant award (to G. G. H.) and by National Institutes of Health Grants DK-45817 and DK-52166 (to G. G. H.) and DK-30834 and DK-30457 (to J. F. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Present address: Dept. of Physiology and Neuroscience, New York University Medical Center, New York, NY 10016. Tel.: 212-263-5434; Fax: 212-689-9060; E-mail: holz{at}helix.mgh.harvard.edu.
** Present address: Hagedorn Research Inst., Niels Steensensve 6, DK 2820 Gentofte, Denmark.
Investigator of the Howard Hughes Medical Institute.
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ABBREVIATIONS |
---|
The abbreviations used are: GLP-1, glucagon-like peptide-1; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-polymerase chain reaction; PKA, protein kinase A; BODIPY FL-X, 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino) hexanoic acid; CICR, Ca2+-induced Ca2+ release; RYR, ryanodine receptor; FBS, fetal bovine serum; SERCA, sarco(endo)plasmic reticulum Ca2+-ATPase; VDCC, voltage-dependent Ca2+ channel; IP3, inositol trisphosphate; cADPR, cyclic ADP-ribose; K-ATP, ATP-sensitive K+ channel; 8-Br-cAMP, 8-bromo-cAMP; bp, base pair(s); SES, standard extracellular salt solution.
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REFERENCES |
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