 |
INTRODUCTION |
Increases in cytoplasmic Ca2+ concentration
([Ca2+]c)1
are important for stimulation of neurosecretion in general (1) and for
the activation of insulin secretion from pancreatic islet
-cells (1,
2). In the latter cell type, increases in [Ca2+]c
usually occur as a result of either nutrient-induced influx of
Ca2+ ions through voltage-gated Ca2+ channels
on the plasma membrane (3) or via the release of Ca2+ from
intracellular Ca2+ stores (4). The endoplasmic reticulum
(ER) (5, 6) and Golgi apparatus (7) probably represent the major
Ca2+ stores in
-cells (8-10) as in other cell types
(11). However, we have recently provided evidence that dense core
secretory vesicles also play a role in intracellular Ca2+
signaling in
-cells (9, 12). Importantly, secretory vesicles occupy
a substantial proportion of the intracellular volume of
-cells (13,
14) and may contain close to half the total cellular Ca2+.
As such, these organelles potentially provide a huge store of mobilizable Ca2+ ions (15).
Previous studies involving measurements of intravesicular free
Ca2+ concentration ([Ca2+]SV) and
immunoelectron microscopy (9) indicated that ryanodine, but not
inositol 1,4,5-trisphosphate (11), receptors mediate Ca2+
release from secretory vesicles in
-cells (9, 10, 16). cDNAs
encoding three RyR isoforms have so far been identified in mammals. The
type I isoform (RyR I) is expressed mainly in skeletal muscle (17),
whereas RyR II is abundant in the heart (18). RyR III is present in a
variety of tissues and cell types, most notably the brain (19). RyR II
has been reported previously to be the most abundantly expressed
isoform (at the mRNA level) in wild type (20) and ob/ob
mouse islets (20, 21), as well as in rat islets (22) and clonal
TC3
cells (22). Moreover, the presence of RyR II protein has also been
demonstrated in derived INS-1
-cells (23). Lower levels of RyR I and
RyR III mRNA have also been detected in
TC3 (22) and HIT-T15
cells (24), respectively. However, the physiological role(s) of RyRs in
-cells remains unclear, given that RyR II mRNA levels in
ob/ob mouse islets are reportedly ~1000-fold less than in
the heart (21), whereas RyR II protein levels in INS-1
-cells were
~10-fold lower than in brain (23).
Receptors for nicotinic acid adenine dinucleotide phosphate (NAADP), a
novel intracellular Ca2+-mobilizing agent (25), may
represent an alternative pathway for Ca2+ efflux from dense
core secretory vesicles (26). Although other studies (27-30) have
demonstrated NAADP-induced Ca2+ release in a variety of
mammalian cells and cell lines, few data are currently available
regarding the role of NAADP in the
-cell. Although functional
NAADP-sensitive Ca2+ stores were recently revealed in human
-cells (31), NAADP-induced Ca2+ release was not observed
in dispersed
-cells from either normal or ob/ob mouse
islets (32). However, in the latter report, neither the RyR agonists
caffeine and ryanodine nor cyclic ADP-ribose (cADPr) induced
Ca2+ release.
In the present study, we show that islets and MIN6
-cells express
two RyR isoforms, RyR I and RyR II, that display distinct subcellular
localizations. Thus, whereas type I RyRs are present at approximately
equal density in a vesicle/mitochondrial fraction, and in microsomes,
RyR II was considerably more abundant on ER membranes. Surprisingly,
dantrolene, a selective inhibitor of RyR I, increased steady-state free
[Ca2+] in secretory vesicles but not in the ER,
suggesting the presence on vesicles of a further activator or channel
capable of amplifying the effects of RyRs on Ca2+ release.
We provide evidence that receptors for NAADP may serve this role, and
we thus demonstrate that secretory vesicles, but not the ER, are an
NAADP-responsive Ca2+ store in
-cells.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Adenoviral Infection--
MIN6 cells, a well
differentiated mouse insulinoma
-cell line (33) (passages
20-30), were grown in Dulbecco's modified Eagle's medium
(Invitrogen) containing 25 mM glucose and 2 mM pyruvate, and supplemented with 15% (v/v) fetal bovine serum, 20 mM glutamine, 100 units/ml penicillin, 100 µg/ml
streptomycin, and 50 µM
-mercaptoethanol, in a
humidified atmosphere containing 5% CO2 (9, 34). For
[Ca2+] measurements with recombinant aequorins (see
below), cells were seeded onto 13-mm diameter
poly-L-lysine-coated glass coverslips and grown to 50-80%
confluency. Cells were then infected with Cyt.Aq, VAMP.Aq, or ER.Aq
adenoviruses encoding untargeted aequorin (Cyt.Aq) (35) or aequorin
targeted to either the secretory vesicles (VAMP.Aq) (9) or the ER
(ER.Aq) (36), at a multiplicity of infection of 30 infectious units per
cell. Measurements of aequorin bioluminescence were performed using a
purpose-built photomultiplier system 48 h after infection, as
described previously (9, 37).
Detection of mRNA for Ryanodine Receptors in
-Cells and
Islets--
Total RNA was extracted from cell lines or rat tissue
using TRI ReagentTM (Sigma) according to the
manufacturer's instructions and reverse-transcribed using Moloney
murine leukemia virus-reverse transcriptase (Promega). PCR
amplification was performed with primers designed to amplify an
isoform-specific region of each of the three RyR subtypes (38) as
follows: RyR I (forward, 5'-GAAGGTTCTGGACAAACACGGG-3'; reverse, 5'-TCGCTCTTGTTGTAGAATTTGCGG-3'); RyR II (forward,
5'-GAATCAGTGAGTTACTGGGCATGG-3'; reverse,
5'-CTGGTCTCTGAGTTCTCCAAAAGC-3'); and RyR III (forward, 5'-CCTTCGCTATCAACTTCATCCTGC-3'; reverse,
5'-TCTTCTACTGGGCTAAAGTCAAGG-3'). The PCR mix consisted of 5 µl
of 10× Buffer 3 (Roche Molecular Biochemicals), 6 µl of 25 mM MgCl2, 1 µl of 10 mM dNTPs,
0.25 µl of Taq DNA polymerase (Roche Molecular
Biochemicals), 5 µl of reverse transcriptase-PCR
cDNA, 0.4 µM forward primer, 0.4 µM
reverse primers, and distilled H2O, at a final volume of 50 µl. Amplification conditions are as follows: 94 °C for 2 min and then 94 °C for 45 s, 55.5 °C for 45 s, 72 °C for 1 min for 32 cycles and then 72 °C for 10 min. Negative controls were
performed by omission of the reverse transcription step or by exclusion of the template from the PCR. PCR products were separated by migration on a 2% (w/v) agarose gel. Products were excised and purified using a
QIAQuickTM gel extraction kit (Qiagen) and subjected to
restriction digest analysis and automated sequencing.
Semi-quantitative PCR--
Total RNA from rat islets, skeletal
muscle, or heart was reverse-transcribed, and semi-quantitative PCR was
performed in the following mix: 5 µl of 10× Buffer 3 (Roche
Molecular Biochemicals), 6 µl of 25 mM MgCl2,
1 µl of 10 mM dNTPs, 0.25 µl of Taq DNA
polymerase (Roche Molecular Biochemicals), 5 µl of reverse
transcriptase-PCR cDNA, 0.4 µM RyR I or RyR II
primers, 0.1 µM
-actin primers (39), and distilled
H2O at a final volume of 50 µl. Semi-quantitative PCR was
then performed as follows: RyR I primers, 94 °C for 2 min and then
94 °C for 45 s, 55 °C for 45 s, 72 °C for 1 min, for
25 cycles and then 72 °C for 10 min; RyR II primers, 94 °C for 2 min and then 94 °C for 45 s, 58 °C for 45 s, 72 °C
for 1 min, for 20 cycles and then 72 °C for 10 min. Amplification in the linear phase was confirmed in each case by trials with 12-30 cycles (data not shown).
Preparation of Cell Lysates and Membrane Fractions--
MIN6
cells or homogenized rat skeletal muscle were extracted into
radioimmunoprecipitation assay (RIPA) buffer, consisting of
phosphate-buffered saline supplemented with 1% (v/v) Nonidet P-40,
0.5% (w/v) sodium deoxycholate, and 0.1% (w/v) SDS. Secretory vesicle
protein was obtained by immunoadsorption of phogrin.EGFP-containing vesicles (40). In brief, MIN6 cells infected with phogrin.EGFP were
homogenized, and the nuclear fraction was removed by centrifugation. Pre-cleared homogenate was then incubated with anti-GFP antibody (Roche
Molecular Biochemicals) bound to protein A-Sepharose. Immunoadsorbed vesicles were washed then incubated at 99 °C for 10 min in SDS-PAGE loading buffer to dissociate vesicle proteins.
To generate MIN6 membrane fractions, cells were harvested in
Tris-saline buffer (10 mM Tris-HCl, 0.14 M
NaCl, pH 7.4) containing 5% (v/v) Tween 40. Fractions were collected
by differential centrifugation using the following spins: 200 × g for 10 min, nuclear fraction; 15,000 × g
for 5 min, mitochondrial/secretory vesicle fraction; 100,000 × g for 1 h, plasma membrane, ER, Golgi fraction;
supernatant from 100,000 × g spin, cytosolic fraction.
Ryanodine Receptor Type I Antiserum Production and
Purification--
Rabbit polyclonal antiserum was raised to a RyR
I-specific sequence of 15 amino acids (residues 830-845;
RREGPRGPHLVGPSRC) that is 100% conserved in all known mammalian RyRI
sequences and absent from the mammalian RyR II and III sequences.
Rabbits (New Zealand White) were immunized with the keyhole limpet
hemocyanin-conjugated peptide as described previously (41), and
antibody specificity was confirmed by enzyme-linked immunosorbent assay
and immunoblot analysis with brain, skeletal, and cardiac muscle
microsomes, prepared as described previously (41). For immunoblot
analysis, microsomes were separated on a 5% (v/v) polyacrylamide gel
(30 µg of protein/lane), and proteins were electrophoretically
transferred to polyvinylidene difluoride membrane before probing with
antibody at a dilution of 1:1000. Affinity-purified antibody (anti-RyR I; number 2142) was prepared by acid elution following incubation of
the crude RyR I antisera either with skeletal muscle RyR protein immobilized on polyvinylidene difluoride membrane strips or on protein
A-agarose columns (Sigma).
Immunoblotting--
Protein samples were resolved by SDS-PAGE on
5% (v/v) polyacrylamide gels and transferred onto Immobilon-P transfer
membrane (Millipore) following a standard protocol. Membranes were
probed with anti-skeletal muscle RyR antibody (1:2500; Upstate
Biotechnology, Inc.), anti-RyR I (1:500; number 2142), anti-RyR II
(1:200; Affinity BioReagents), and anti-RyR III antibodies (1:500;
number 110E) (42). Immunostaining was revealed with horseradish
peroxidase-conjugated anti-rabbit IgG (Sigma; 1:100 000), anti-mouse
IgG (Sigma; 1:10,000), or anti-sheep IgG (Dako; 1:4000) using an
enhanced chemiluminescence (ECL) detection system (Roche Diagnostics).
Measurements of Free Ca2+ Concentration with
Recombinant Targeted Aequorins--
Cells were depleted of
Ca2+ by incubation with ionomycin (10 µM),
monensin (10 µM), and cyclopiazonic acid (10 µM) in modified Krebs-Ringer bicarbonate buffer (KRB: 140 mM NaCl, 3.5 mM KCl, 0.5 mM
NaH2PO4, 0.5 mM MgSO4,
3 mM glucose, 10 mM Hepes, 2 mM NaHCO3, pH 7.4) supplemented with 1 mM EGTA,
for 10 min at 4 °C (9). Aequorin was reconstituted with 5 µM coelenterazine n (43) for 1-2 h at 4 °C
in KRB supplemented with 1 mM EGTA.
Intact cells were perifused with KRB plus additions as stated at 2 ml·min
1 in a thermostatted chamber (37 °C) in close
proximity to a photomultiplier tube (ThornEMI) (44). Where indicated,
cells were permeabilized with 20 µM digitonin for 1 min
at 37 °C and subsequently perifused in intracellular buffer (IB: 140 mM KCl, 10 mM NaCl, 1 mM
KH2PO4, 5.5 mM glucose, 2 mM MgSO4, 1 mM ATP, 2 mM sodium succinate, 20 mM Hepes, pH 7.05).
Additions to this buffer were as stated in the figure legends. At the
end of all experiments cells were lysed in a hypotonic
Ca2+-rich solution (100 µM digitonin, 10 mM Ca2+ in H20) to discharge the
remaining aequorin pool for calibration of the aequorin signal (9).
Microinjection, Flash Photolysis, and Ca2+
Imaging--
MIN6 cells were seeded onto 24 mm
poly-L-lysine-coated coverslips and microinjected, as
previously described (45-47), with Oregon Green 488 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-
tetraacetic acid-1 dextran (Molecular Probes, Eugene, OR; 2.5 mg·ml
1) in Tris-HCl buffer, pH 8.0, in the absence
(control) or presence of 2 µM caged NAADP (48) (kindly
provided by Dr. Luigia Santella, University of Naples). The
concentration of caged NAADP in the microinjection pipette was 2 µM, resulting in a final intracellular concentration of
50-150 nM, given an injection volume of 2.5-7.5% of the
total cell volume (49). 2-4 h after microinjection, cells were imaged
on a Leica TCS-SP2 laser-scanning confocal illumination system attached
to a Leica DM IRBE inverted epifluorescence microscope, using a 63× PL
Apo 1.4 numerical aperture oil immersion objective. Fluorescence was
excited at 488 nm (argon laser), and fluorescence was detected at
wavelengths longer than 515 nm using a long pass cut-off filter at this
wavelength. A second laser (CoherentTM) provided light for
1 s at wavelengths of 351 and 364 nm (via the objective lens) and
was used for photolysis of caged NAADP at selected regions of interest
within the cell (corresponding to ~1% of the surface of the confocal
slice). Images were acquired at 5-s intervals before and after the UV
pulse. Where indicated, ER/Golgi Ca2+ stores were depleted
of Ca2+ by washing the cells in Ca2+-free KRB
and incubation in Ca2+-free KRB supplemented with 1 µM thapsigargin, 10 µM cyclopiazonic acid,
and 1 mM EGTA for 10 min. Cells were then maintained in Ca2+-free KRB during confocal imaging and photorelease of
NAADP.
Assay of Insulin Secretion--
MIN6 cells were incubated in
full growth medium (see above) containing 3 mM glucose for
16 h and then incubated in KRB supplemented with 3 mM
glucose for 30 min at 37 °C. The medium was removed and retained,
and cells were then stimulated with KRB supplemented with 30 mM glucose for a further 30 min at 37 °C. Released and total insulin were measured by radioimmunoassay (34).
Statistics--
Free [Ca2+] was calculated using
METLIG software (50). Data represent the means ± S.E. of at least
three separate experiments. Statistical analysis was performed using
the paired Student's t test.
 |
RESULTS |
Detection of Ryanodine Receptor mRNAs in
-Cell Lines and
Primary Islets--
Using isoform-specific primers, PCR products
corresponding to RyR I and RyR II cDNA were readily amplified from
MIN6-, INS1-, or rat islet-derived cDNAs (data not shown). By
contrast, RyR III cDNA was not amplified from these sources using
the chosen primer pair (see "Experimental Procedures"), although
RyR III from brain cDNA was amplified, as expected. The identity of
each of the generated PCR products was confirmed by both restriction analysis and by automatic sequencing, which revealed 100% identity with the corresponding mouse (GenBankTM accession
numbers X83932 and AF295105 for RyR I and RyR II, respectively) and rat
(GenBankTM accession numbers AF130879 and U95157)
cDNAs. Semi-quantitative PCR revealed ~5-fold lower RyR I
mRNA levels in rat islet-derived than in skeletal muscle-derived
cDNA, and RyR II mRNA levels were ~8-fold lower in islet than
in heart cDNA (data not shown).
Expression of Ryanodine Receptor Protein in MIN6 Cells--
In
order to confirm the presence, and identify the intracellular
localization, of RyRs in MIN6
-cells, subcellular fractionation and
immunoblotting (Western) was performed. Probing of crude MIN6 cell
fractions with a subtype-specific RyR antibody, which recognizes type I
(and III) isoforms, indicated similar levels of RyR immunoreactivity in
both mitochondrial/dense core secretory vesicle and microsomal fractions (Fig. 1A,
upper panel). By contrast, RyR II immunoreactivity was much
more abundant on the latter fraction, with only weak staining for RyR
II in the crude vesicle/mitochondria fraction (Fig. 1A,
lower panel). To demonstrate that RyR I immunoreactivity was
present on secretory vesicles in the crude secretory
vesicle/mitochondrial fraction examined above, immunoblotting was also
performed with immunopurified dense core secretory vesicles (40).
Immunoreactivity toward both anti-RyR I/III, and to a selective
anti-RyR I antibody (see "Experimental Procedures"), was clearly
evident in these membranes (Fig. 1B, upper
panels), whereas reactivity to RyR III (Fig. 1B,
lower panel) was undetectable.

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Fig. 1.
Immunoblot of RyRs in MIN6
-cells. A, subcellular
fractions of MIN6 cells obtained by differential centrifugation were
migrated on a 5% (v/v) polyacrylamide gel, transferred onto an
Immobilon-P transfer membrane, and stained with anti-skeletal muscle
RyR (type I) or anti-RyR II antibody. The latter antibody is known to
also weakly react with RyR I. Lanes were loaded as follows: lane
1, whole cell lysate; lane 2, cytosolic fraction;
lane 3, nuclear fraction; lane 4, mitochondria
and secretory vesicles; lane 5, ER and Golgi.
B, whole cell lysate from MIN6 cells (lane
1), skeletal muscle protein (lane 2), and MIN6 cell
vesicle protein (see "Experimental Procedures"; lane 3)
were separated on a 5% (v/v) polyacrylamide gel, transferred onto a
membrane, and probed with anti-skeletal muscle RyR antibody or
isoform-specific antibodies, RyR I and RyR III.
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Effect of Ryanodine Receptor Inhibition on Secretory Vesicle and ER
Ca2+ Concentrations--
The above fractionation studies
indicated that the relative abundance of type I/type II RyRs was higher
in secretory vesicles than the ER but provided no information on the
relative abundance of the two isoforms on either membrane, considered
alone. To determine the relative importance of RyR I and RyR II on each
organelle, we therefore used a functional approach in living MIN6
cells. Recombinant aequorins, targeted specifically to either organelle by the addition of appropriate presequences (9), were utilized to
monitor free Ca2+ concentrations in each compartment.
Concentrations of ryanodine (10 µM) expected to lead to
closure of all RyR isoforms (51) substantially raised the steady-state
concentrations of free Ca2+ in both the secretory vesicle
matrix ([Ca2+]SV) and the ER lumen
([Ca2+]ER) (Fig.
2, A and B). In
contrast, the skeletal muscle relaxant dantrolene (52) significantly
increased [Ca2+]SV (from 40.1 ± 6.7 to
90.4 ± 14.8 µM; n = 4, p < 0.01; Fig. 2C), whereas neither
[Ca2+]ER (Fig. 2D) nor
[Ca2+]c (Fig. 2E) was affected by this
agent. These findings are in agreement with previous studies where
dantrolene was shown to bind directly to and inhibit pig and rabbit
skeletal muscle (types I and III), but not cardiac (type II), RyRs
(52-55), suggesting that dantrolene inhibits the type I, but not the
type II, RyR in MIN6
-cells.

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Fig. 2.
Effect of ryanodine and dantrolene on
steady-state Ca2+ concentrations in the secretory vesicle
and ER. After Ca2+ depletion and aequorin
reconstitution, MIN6 cells infected with VAMP.Aq (A and
C), ER.Aq (B and D), or Cyt.Aq
(E) encoding adenoviruses were perifused in KRB containing 1 mM EGTA. Where indicated, EGTA was replaced with 1.5 mM CaCl2 in the absence (open
symbols) or presence (closed symbols) of ryanodine
(Ryan.) (10 µM; A and B)
or dantrolene (Dan.) (10 µM; C-E).
Data are the means of four separate experiments.
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Effect of NAADP on Secretory Vesicle and ER Ca2+
Concentrations--
The above results suggest that although
ryanodine-sensitive Ca2+ efflux from secretory vesicles is
likely to be mediated principally via type I RyRs, this channel subtype
apparently plays a minor role, if any, in mediating Ca2+
release from the ER in MIN6
-cells. This result was unexpected given
that subcellular fractions enriched with ER membranes apparently contained the same amount or more immunoreactivity to RyR I as a crude
secretory vesicle/mitochondria fraction (Fig. 1A,
upper panel). One simple explanation of this observation is
that the absolute number of type II RyRs on the ER is very much greater than type I receptors, a difference that may not be apparent given the
different antibodies and dilutions used to quantitate each of these
isoforms (Fig. 1).
However, as an alternative explanation, we next explored the
possibility that another Ca2+ release channel may be
functional on secretory vesicles, whose presence may stimulate the
activity of neighboring RyR I channels. The effects on
[Ca2+]SV of the recently identified
Ca2+-mobilizing molecule NAADP (25, 56) were therefore
examined in permeabilized cells at a concentration of this compound
previously shown to be optimal in human
-cells (31) and other
mammalian cell types (26). 100 nM NAADP caused a small but
highly significant decrease in [Ca2+]SV (Fig.
3A) but was completely without
effect on [Ca2+]ER (Fig. 3B).

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Fig. 3.
Effect of NAADP on secretory vesicle and ER
Ca2+ concentrations in permeabilized MIN6 cells.
After Ca2+ depletion and aequorin
reconstitution, MIN6 cells infected with VAMP.Aq
(A and C-E) or ER.Aq (B) encoding
adenoviruses were permeabilized and perifused in IB containing 1 mM EGTA. Where indicated, free [Ca2+] was
increased from <1 to 400 nM using an EGTA-buffered
Ca2+ solution (see "Experimental Procedures"). Cells
were perifused with 100 nM NAADP at steady-state
[Ca2+]. Ryanodine (C), dantrolene
(D), or nimodipine (E) were present throughout
the experiment. Data are means of four separate experiments.
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To determine whether the effects of NAADP may be mediated by a receptor
identical or similar to that described previously (26) in mammalian and
other cell systems, we next explored the pharmacology of the observed
NAADP-induced changes in secretory vesicle [Ca2+].
Concentrations of ryanodine sufficient to inhibit all RyR isoforms (51), but known to have no effect on NAADP receptor activity (57),
failed to alter NAADP-induced [Ca2+]SV
changes (Fig. 3C). Similarly, NAADP-induced release of
secretory vesicle Ca2+ was unaffected by dantrolene (Fig.
3D). By contrast, nimodipine, an inhibitor of L-type
Ca2+ channels shown previously (26) to block NAADP
receptors, completely blocked NAADP-induced changes in
[Ca2+]SV (Fig. 3E).
Effect of Photorelease of Caged NAADP on
[Ca2+]c in Intact MIN6 Cells--
To determine
whether (i) NAADP may mediate Ca2+ release selectively from
dense core vesicles in living cells, and (ii) to explore the impact of
this release of cytosolic Ca2+ concentrations, we next
micro-injected an inactive precursor of NAADP ("caged NAADP") (48)
into single MIN6
-cells, and we monitored the impact of its rapid
uncaging by flash photolysis (Fig. 4).
Photo-released NAADP provoked an increase in the fluorescence of the
co-microinjected Ca2+ reporter, Oregon Green, of 4.3 ± 2.1% with respect to basal fluorescence (n = 7 cells; Fig. 4A), consistent with the mobilization of
intracellular Ca2+. The magnitude of this increase was not
significantly affected by depletion of ER/Golgi Ca2+ stores
with the sarco(endo)plasmic reticulum Ca2+-ATPase
inhibitor, thapsigargin (5.6 ± 1.4%; n = 8 cells; Fig. 4B), nor by incubation with ryanodine (3.7 ± 0.9%; n = 7 cells; Fig. 4C).

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Fig. 4.
Effect of uncaging NAADP on cytosolic
Ca2+ concentrations in single living MIN6 cells.
A, cells were microinjected with Oregon Green 488 bis(2-aminophenoxy)ethane-N,N,N',N'-
tetraacetic acid-1 dextran in the absence (control) or presence of 2 µM caged NAADP. Confocal images were acquired at 5-s
intervals before and after induction of photolysis with UV light
(~360 nm) for 1 s. Microinjected cells were incubated with
ryanodine (10 µM; C) or depleted of
Ca2+ (see "Experimental Procedures") and incubated with
thapsigargin (1 µM; B) for 5 min prior to
imaging and photolysis. Data represent the means of normalized
fluorescence values for at least seven cells in each case.
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Importance of Secretory Vesicle Ca2+ Release for
Glucose-stimulated Insulin Secretion--
The above studies indicated
that release of Ca2+ from secretory vesicles, mediated by
either type I RyR or NAADP receptors, may be important for the
triggering of insulin secretion by nutrients. To determine whether
Ca2+-induced Ca2+ release via type I RyRs or
Ca2+ release through NAADP receptors was qualitatively the
more important pathway for Ca2+ efflux in living cells, we
blocked the former with dantrolene (see above). In accordance with
previous findings in islets (58) and INS1E cells (59), the stimulation
of insulin release from MIN6
-cells by 30 mM
(versus 3 mM) glucose (3.07 ± 0.51-fold; n = 3, p < 0.01)
was completely inhibited in the presence of the drug (Fig.
5A). Similarly, glucose-induced insulin secretion was abolished (from 2.02 ± 0.11-fold stimulation to no significant effect; n = 4, p < 0.01) in the
presence of 100 µM ryanodine (Fig. 5B). This
latter finding is in contrast to previous reports where ryanodine had
little or no effect on insulin secretion in mouse
-cells (60) nor in
INS1E cells (59), a result attributed to the poor permeation of
ryanodine across the cell membrane in these systems. By contrast, both
the present (Fig. 2, A and B) and previous
studies (61) indicate that intact MIN6 cells may be more permeable to
this drug.

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Fig. 5.
Dantrolene and ryanodine inhibit
glucose-induced insulin secretion. MIN6 cells were cultured for
16 h with 3 mM glucose and then incubated with KRB
supplemented with 3 mM glucose for 30 min. Cells were then
incubated for a further 30 min with 3 or 30 mM glucose,
with additions as stated. Released insulin was measured by
radioimmunoassay (see "Experimental Procedures"). Data are the
means of at least three separate experiments.
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 |
DISCUSSION |
Ryanodine Receptor mRNA Expression in Pancreatic
-Cells--
In this study, we found that mRNAs encoding both
RyR I and RyR II are expressed in
-cell lines as well as in rat
islets at approximately equal levels. These findings are in apparent
contrast with previous reports (20-22) indicating that RyR II mRNA
was by far the most abundant RyR isoform, at least in mouse
-cells. Thus, in these earlier studies, only faint reverse transcriptase-PCR products were generated with RyR I primers in mouse-derived
TC3 cells, whereas the same primers did not detect any RyR I mRNA in
rat islets (22). Similarly, ob/ob mouse islets, which are enriched in
-cells, were shown to contain ~1000-fold less RyR II
mRNA than the heart (21). A number of factors may underlie these
apparently discrepant results. First, the primers used to detect RyR I
in previous reports may have been less efficient in amplifying the
-cell RyR I than those used in the present study. Consistent with
this view, the primers used by Takasawa et al. (20) scarcely
detected RyR I mRNA in the brain, a tissue in which this isoform is
abundant (19, 62). Second, the presence and density of RyRs in
different
-cell lines and rodent strains may well differ. Thus, RyR
II protein was hardly detectable in RINm5F cells, whereas INS1 cells
were shown to express significant amounts (23). Similarly, one group
(21) described low levels of RyR II mRNA in ob/ob mice
islets, whereas another (20) failed to detect this message in islets
from distinct colonies of ob/ob mice. An intriguing
possibility is that these differences in RyR II expression in the islet
may contribute to the differing severities of diabetes in the different
mouse strains.
Ryanodine Receptor Protein Expression and Subcellular Localization
in MIN6
-Cells--
RyR subtypes are expressed in various
combinations in specific tissues and cell types. Thus, RyR II and III
are expressed in the heart, RyR I and RyR III in skeletal muscle, and
all three subtypes in smooth muscle and brain (62). Although the
relevance of multiple isoform expression is not clearly understood, one possibility is that co-expression serves to amplify Ca2+
signals. Indeed, such a mechanism may explain why the presence of both
RyR I and RyR II is required for the activation of
Ca2+-induced Ca2+ release upon Ca2+
influx in vascular myocytes (63).
The present study revealed the presence of both RyR I and RyR II in
MIN6
-cells and islets at both the mRNA and protein levels (Fig.
1). Moreover, using subcellular fractionation as well as vesicle
immunopurification, we demonstrate a distinct subcellular localization
for each RyR isoform in this cell type. Thus, type I RyRs were present
on both the ER and secretory vesicles, whereas RyR II immunoreactivity
was more abundant in the former. Although we also detected a small
amount of RyR II immunoreactivity in the crude mitochondrial/secretory
vesicle fraction (Fig. 1A), it should be noted that at least
part of this reactivity may result from contamination of this fraction
with ER/Golgi fragments and/or cross-reactivity with RyR I, because the
anti-type II RyR antibody used also weakly cross-reacts with RyR I.
Supporting the view that Ca2+-induced Ca2+
release from secretory vesicles is mediated principally via RyR I
channels, and from the ER via RyR II, blockade of RyR I receptors in
whole cells with dantrolene (55) affected steady-state Ca2+
concentrations only in the former compartment (Fig. 2, C
versus D). Together, these data therefore provide
both structural and functional evidence that RyR I and RyR II are
located on distinct organelles in MIN6
-cells.
Interestingly, inhibition of type I RyRs, shown here (Fig. 5) and in
earlier studies (58) to block glucose-induced insulin secretion, now
seems likely to involve largely a blockade of Ca2+ release
from secretory vesicles, rather than from the ER (Fig. 2). This result,
which accords well with the reported effects of depleting vesicle
Ca2+ on insulin release (64), reinforces the view that the
release of vesicle Ca2+ plays an important role in
triggering or facilitating the exocytotic release of insulin (Fig.
6).

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Fig. 6.
Potential cross-talk between two different
but converging messenger pathways that lead to Ca2+ release
from dense core secretory vesicles. Exposure of islet -cells or
MIN6 cells to nutrients causes an increase in intracellular free
[ATP], which in turn may modulate the activity of CD38, increasing
the intracellular concentrations of NAADP and cADPr. NAADP-induced
Ca2+ release from the secretory vesicles (1)
could potentially activate Ca2+-induced Ca2+
release from the neighboring type I RyRs (2), previously
sensitized to Ca2+ by cADPr (3). This would
result in a domain of high Ca2+ concentration underneath
the plasma membrane, independent of Ca2+ influx through
voltage-gated Ca2+ channels, which may contribute to the
stimulation of insulin secretion.
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Role of NAADP Receptors in
-Cells--
An unexpected finding of
the present study was that dantrolene, which blocks RyR I channels,
affected Ca2+ concentrations in secretory vesicles but not
in the ER despite the presence of RyR I receptors on both organelles.
One possible explanation for this result is our demonstration that MIN6
-cells possess an NAADP-sensitive intracellular Ca2+
store that appears to coincide, at least in part, with secretory vesicles (Figs. 3 and 4). Importantly, these data are consistent with
previous findings that have suggested NAADP releases Ca2+
from a non-ER Ca2+ pool at the frog neuromuscular junction
(65) and that the NAADP-sensitive Ca2+ pool in sea urchin
eggs is thapsigargin-insensitive (66).
We show here that in pancreatic
-cells, NAADP-induced
Ca2+ release is insensitive to ryanodine and dantrolene,
confirming previous reports (26) that NAADP acts on a channel distinct
from the RyR (Figs. 3, C and D, and
4C). Although the receptor for NAADP is as yet unidentified
in molecular terms, in agreement with previous findings in sea urchin
eggs (67), brain (28), smooth muscle (68), and heart (57), L-type
Ca2+ channel inhibitors were found here to block
NAADP-induced Ca2+ release from
-cell vesicles (Fig.
3E), suggesting that a common or similar receptor is
involved in each of these cellular systems. Interestingly,
NAADP-mediated Ca2+ release in living
-cells was not
significantly affected by blockade of RyRs (Fig. 4, C
versus A), a result also consistent with an action of this messenger via a non-RyR channel.
Two Converging Pathways Are Involved in Ca2+ Release
from Secretory Vesicles--
cADPr, the proposed endogenous ligand for
RyRs (69, 70), and NAADP are likely to be synthesized in
-cells by
the same enzyme, an ADP ribosyl cyclase termed CD38 (71). At the cell surface, the catalytic domain of CD38 is likely to be exposed to the
extracellular space (72). However, internalized catalytically active
CD38 is found in non-clathrin-coated vesicles (73). Moreover, endocytotic vesicles have been shown previously to accumulate the
precursor for cADPr,
-NAD, via an unidentified dinucleotide transport system (74). As well as catalyzing the conversion of
-NAD
to cADPr, CD38 itself is thought to be involved in pumping the cyclic
nucleotide into the cytosol (75), thus providing a potential mechanism
for the accumulation of intracellular cADPr and potentially NAADP.
Interestingly, transgenic mice overexpressing CD38 specifically in
-cells show enhanced glucose-induced insulin secretion (76), whereas
deletion of both alleles of CD38 gives rise to glucose intolerance
(77). Because CD38 activity is regulated allosterically by ATP (76),
glucose-induced increases in the intracellular free concentration of
this nucleotide (47, 78) may lead to increases in the intracellular
concentrations of either cADPr, NAADP, or both. Although
glucose-dependent increases in cADPr have been reported
previously (69), key future goals will be to determine whether glucose
is able to increase NAADP levels in
-cells and whether such
increases are altered in models of type 2 diabetes mellitus.