1 Molecular Medicine Section, Department of Neuroscience, University of Siena,
via Aldo Moro 5, 53100 Siena, Italy
2 Laboratory of Molecular Signalling, The Babraham Institute, Babraham,
Cambridge CB2 4AT, UK
3 Department of Anesthesia Brigham and Women's Hospital, Boston, MA 02115,
USA
* Author for correspondence (e-mail: v.sorrentino{at}unisi.it )
Accepted 18 March 2002
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Summary |
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Key words: Ryanodine receptor channels, Calcium signalling, Calcium stores, Endoplasmic sarcoplasmic reticulum, Calcium release
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Introduction |
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We report here on experiments with human HEK 293 cells transfected with either the RyR type 1 (RYR1) or the type 3 (RyR3) isoforms. Since epithelial HEK 293 are not a cell type specialised for RyR-mediated Ca2+ release, we reasoned that they might represent a good model to study the localisation and the activity of RyR channels in a non-specialised endoplasmic reticulum (i.e. in contrast to the sarcoplasmic reticulum). In addition, HEK 293 cells should provide a simpler environment to compare functional properties and dynamics of Ca2+ release of different isoforms of RyRs expressed under the same cellular context, independently of the variable number of accessory proteins.
We have found that heterologous RyR1 and RyR3 channels are homogeneously distributed in the endoplasmic reticulum of HEK 293 cells, at least at the level of resolution of confocal microscopy. Functionally, the transfected channels, when stimulated with caffeine, are able to release Ca2+ with a global response involving the entire endoplasmic reticulum. In contrast, expression of recombinant RyR3 channels, but not of RyR1 channels, results in the appearance of spontaneous and localised Ca2+-release activity in HEK 293 cells. At the same time, spontaneous Ca2+-release activity in RyR3-expressing HEK 293 cells is restricted to one or two sites, indicating that preferential domains for generating localised Ca2+ release events are present in these cells.
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Materials and Methods |
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Cell culture and transfection
Human embryonic kidney (HEK 293) cells were maintained in -MEM
medium supplemented with 2 mM glutamine (Bio-Wittaker, Walkersville, MD), 100
µg/ml streptomycin, 100 U/ml penicillin (Bio-Wittaker), 1 mM sodium
pyruvate (Bio-Wittaker), 10% heat-inactivated fetal calf serum (FCS)
(Bio-Wittaker) at 37°C under 5% CO2. DNA transfection of the
RyR3 expression vector was carried out using the calcium phosphate method.
8x105 cells were plated on a 100 mm tissue culture dish 24
hours before transfection. One hour before DNA addition, the medium was
changed with fresh medium. 10 µg of RyR3 expression vector were mixed in a
solution containing HBS (5 g/l Hepes, 8 g/l NaCl, pH 7.1), 0.7 mM
Na2HPO4, 0.7 mM NaH2PO4, 120 mM
CaCl2 and incubated for 30 minutes. Calcium phosphate precipitates
were added to cells and incubated for 6 hours. Transfections of the
pcDNA3-RyR1 clones were performed with the GenePORTERTM method (Gene
Therapy Systems, San Diego, CA), following the manufacturer's instructions.
For stable transfections, Geneticin sulphate G418 (Life Technology, Groningen,
The Netherlands) was added 48 hours after transfection, at a final
concentration of 800 µg/ml. Single colonies were transferred to a 96-well
multiplate, expanded and tested for RyR expression.
Immunofluorescence staining
Cells were fixed with 3% paraformaldehyde/2% sucrose in PBS (137 mM NaCl,
2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM
KH2PO4) for 5 minutes at room temperature, washed, and
incubated for 15 minutes in 2% BSA or 5% goat serum in PBS. After washing,
cells were permeabilised with Hepes Triton Buffer (20 mM Hepes pH 7.4, 300 mM
sucrose, 50 mM NaCl, 3 mM MgCl2, 0.5% Triton X-100). For RyRs
detection, cells were alternatively incubated overnight at 4°C with a
rabbit polyclonal antibody specific for the RyR1 or the RyR3 isoform at a
1:1000 dilution (Giannini et al.,
1995), or for 1 hour at room temperature with a mouse monoclonal
antibody recognising RyR1 and RyR3 isoforms at a 1:1000 dilution (Alexis
Biochemicals, San Diego, CA). Polyclonal antibodies against calreticulin were
kindly provided by M. Michalak (University of Alberta, Canada) and were used
at a 1:20 dilution for 1 hour at room temperature. Polyclonal antibodies
against Ins(1,4,5)P3R1 were kindly provided by P. De
Camilli (Boyer Centre for Molecular Medicine, Yale University School of
Medicine, New Haven, CT) and was used at a 1:1000 dilution for 1 hour at room
temperature. FITC- and TRITC-conjugated secondary antibodies were from Sigma.
AlexaFluor®-conjugated secondary antibodies were from Molecular Probes
(Eugene, OR). Cy3-conjugated secondary antibodies were from Jackson
ImmunoResearch (West Grove, PA). All secondary antibodies were used
accordingly to the manufacturer's instructions. The expression vector
containing the Green Fluorescent Protein (GFP) cDNA fused to a Golgi targeting
signal was a gift of R. Rizzuto (University of Ferrara, Italy). Images were
collected with an epifluorescence Axioplan 2 imaging Microscope (Zeiss,
Thornwood, NY) equipped with a MicroMAX digital CCD camera (Princeton
Instruments, Trenton, NY), and digitised, stored and subsequently processed
with a Meta Imaging Serie 4.5 software (Universal Imaging Corporation®,
West Chester, PA).
Microsomal proteins preparation
Cells were harvested by a rubber scraper, pelleted in 50 ml tubes and
homogenised in ice cold buffer A (320 mM sucrose, 5 mM Na-Hepes pH 7.4 and 0.1
mM PMSF) using a Teflon potter. Homogenates were centrifuged at 7000
g for 5 minutes at 4°C. The supernatant obtained was
centrifuged at 100,000 g for 1 hour at 4°C. The microsomes
were resuspended in buffer A and stored at -80°C. Protein concentration of
the microsomal fractions was quantified using the Bradford protein assay kit
(Bio-Rad Laboratories, Hercules, CA).
Western blot analysis
Microsomal proteins were separated by SDS-PAGE, as described
(Conti et al., 1996). Proteins
were then transferred to a nitrocellulose membrane (Schleicher & Schuell,
Dassel, Germany) using a transfer buffer containing 192 mM glycine, 25 mM
Tris, 0.01% SDS and 10% methanol for 5 hours at 350 mA at 4°C. Filters
were blocked for 3 hours in a buffer containing 150 mM NaCl, 50 mM Tris-HCl pH
7.4, 0.2% Tween-20, 5% nonfat milk, and incubated overnight at room
temperature with specific antibodies. Rabbit polyclonal antibodies able to
distinguish the three RyRs were used as described
(Giannini et al., 1995
).
Intracellular Ca2+ measurements
Untransfected and transfected HEK 293 cells were loaded with 5 µM Fura
2-AM (Calbiochem® La Jolla, CA) in Krebs-Ringer-Hepes medium (125 mM NaCl,
5 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2 mM
CaCl2, 6 mM glucose and 25 mM Hepes, adjusted to pH 7.4 with NaOH)
for 30 minutes at room temperature in the dark. The Fura 2 fluorescence was
recorded on an inverted stage microscope (Nikon) using a 40x objective.
Fura 2 was excited alternatively at 340 and 380 nm using dual monochromators.
Images were acquired with a digital CCD camera (Princeton Instruments) and
Ca2+ signalling was analysed using computer software (Metafluor,
Universal Imaging Corporation®, West Chester, PA).
Some of the cytosolic Ca2+ measurements were performed as
follows: the culture medium was replaced with an extracellular medium
containing: 121 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl2, 1.8 mM
CaCl2, 6 mM NaHCO3, 5.5 mM glucose, 25 mM Hepes pH 7.3.
Cells were loaded with 1 µM Fura 2-AM or Fluo 3-AM for 45 minutes followed
by a 30 minute de-esterfication period. For video imaging, the coverslips
bearing the Fura-2-loaded cells were mounted on a Nikon Diaphot inverted
microscope and alternately excited with 340 and 380 nm light from twin
monochromators; the excitation wavelengths being switched with a rotating
mirror chopper (Glen Creston Instruments, Stanmore, UK). Emitted light was
filtered at 510 nm and collected with an intensified CCD camera (Photonic
Science, Tunbridge Wells, UK). The video signal was digitised, stored and
subsequently processed off-line with an Imagine image-processing unit
(Synoptics, Cambridge, UK). Ratio images were acquired at 2 second intervals
and [Ca2+]cyt was generated from a modified formula
(Grynkiewicz et al., 1985),
which accounted for signal artefacts
(Bootman and Berridge,
1996
).
Confocal Ca2+ measurements with Fluo-3-loaded cells were
performed as described (Bootman et al.,
1997a), with the modification that images were captured
(256x240 pixels) at an effective rate of 15 frames/second (after
averaging two consecutive images taken at 30 frames/second) using a Noran Oz
laser scanning confocal microscope. For the line scans, a single pixel-wide
line across the image was repetitively scanned at 250 lines/second. The
confocal slit was set to result in a z-section thickness of
1 µm. All
experiments were performed at room temperature (20-22°C). Fluo 3 was
excited using the 488 nm laser line, and the emitted fluorescence was
collected at wavelengths >505 nm. Offline analysis of the confocal data was
performed using a modified version of NIH Image. Absolute values for
Ca2+i were calculated according to the equation:
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[3H]ryanodine-binding assay
Microsomal preparations (30 µg) from control HEK 293, RyR1 and
RyR3-expressing cells were incubated for 1.5 hours at 36°C with 20 nM
[3H]ryanodine in 200 µl of a solution containing 0.2 M KCl, 10
mM Hepes pH 7.4, 10 µM Ca2+ and a mixture of protease
inhibitors: aprotinin (76.8 nM), benzamidine (0.83 mM), iodoacetamide (1 mM),
leupeptin (1.1 mM), pepstatin (0.7 mM) and PMSF (0.1 mM). The bound
[3H]ryanodine was separated from free ligand by filtering through
Whatman GF/B glass fiber microfilters. The filters were washed with 3x5
ml of ice-cold buffer, as described above, and 2x5 ml 10% EtOH.
Radioactivity remaining in the filters was measured by liquid scintillation
counting. Specific binding was calculated as the difference between total and
nonspecific binding measured in parallel assays in the presence of 20 µM
unlabeled ryanodine. All experiments were performed in duplicate.
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Results |
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Intracellular localisation of RyRs in transfected cells
The subcellular distribution of RyR1 and RyR3 in HEK-293-transfected cells
is shown in Fig. 2. Sequential
scanning over an interval ranging from 0.25 to 1 µm showed that RyR1- and
RyR3-specific signals could be detected throughout the endoplasmic reticulum
of transfected cells and that RyRs were distributed as a thin network
spreading as far as the cell periphery. No major differences could be detected
between the distribution of RyR1 and RyR3 proteins in transfected cells.
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To further investigate the subcellular distribution of RyRs,
co-localisation experiments were performed using different markers of the
endoplasmic reticulum. In particular, Fig.
3 shows selected planes displaying the intracellular localisation
of the Ca2+-binding protein calreticulin, the
Ins(1,4,5)P3Rs, the SERCA pumps and the Golgi apparatus
compared with the RyR3 distribution. Experiments performed on RyR1-expressing
cells produced results comparable with those obtained with cells expressing
RyR3 (data not shown). The top panels in
Fig. 3 show the intracellular
distribution of RyR3 compared with that of endogenous calreticulin. In most of
the cells, brighter fluorescence signals were observed in the perinuclear
region of the cell, while a reticular pattern of staining was detected in the
cell periphery. An overlay of representative images from sequential scanning
showed that the patterns of staining obtained with anti-calreticulin and
anti-RyR3 antibodies are extensively superimposable and that they are
consistent with an endoplasmic reticulum distribution of the two proteins. In
addition to RyRs, Ins(1,4,5)P3Rs play a role in the
generation of intracellular Ca2+ signalling. Western blot analysis
and binding experiments with
D-myo-[3H]Ins(1,4,5)P3 performed on microsomes
prepared from RyR-expressing cells and untransfected cells revealed that the
different cell populations express equal amounts of the
Ins(1,4,5)P3R type 1 isoform (data not shown).
Immunolocalisation experiments with antibodies against the
Ins(1,4,5)P3Rs revealed that RyR3 and
Ins(1,4,5)P3R were codistributed in the endoplasmic
reticulum surrounding the nucleus and in the central part of cell, although
some peripheral regions of the cells, negative for RyR staining, were positive
for Ins(1,4,5)P3R immunofluorescence. RyR-expressing cells
were also stained with a monoclonal antibody against SERCA. The SERCA staining
was also compatible, as expected, with a distribution in the endoplasmic
reticulum. Similarly to what was observed for
Ins(1,4,5)P3R, calsequestrin and RyRs, SERCA staining was
more intense in the perinuclear region of the cells, in agreement with a
higher density of endoplasmic reticulum in this region of the cells.
Interestingly, however, SERCA immunostaining revealed a punctuate distribution
of fluorescence, as if there were discrete regions of higher density of SERCA
pumps. Overlay of SERCA images with RyRs pictures revealed only a partial
overlap of the two antigens within the endoplasmic reticulum. Recent work has
revealed that the Golgi apparatus may function as an
Ins(1,4,5)P3-operated Ca2+ store functionally
distinguishable from those of the endoplasmic reticulum
(Pinton et al., 1998). The
subcellular localisation of the Golgi apparatus in RyR-expressing cells was
investigated following cell transfection with plasmids coding for a Green
Fluorescent Protein (GFP) fused to a Golgi targeting signal
(Pinton et al., 1998
). In most
of the cells, the Golgi apparatus appears as a perinuclear convoluted
structure, but no apparent overlay between Golgi apparatus fluorescence signal
and RyR3 staining was observed (Fig.
3), suggesting that RyRs were not enriched in the Golgi, compared
with the endoplasmic reticulum membranes.
|
Intracellular Ca2+ measurements in HEK 293 cells
expressing RyR1 and RyR3
The functional activity of heterologous RyRs expressed in HEK 293 cells was
verified by intracellular Ca2+-release analysis of transfected
cells. Cells were stimulated with caffeine, an activator of RyR
Ca2+-release channels and analysed by video imaging of
Fura-2-loaded cells. Caffeine solutions were applied to cells using either
bulk solution changes or using a continuous gravity-driven superfusion.
Neither of these methods evoked a change in Ca2+ levels. This
differs from data reported by Querfurth et al., who found expression of low
levels of endogenous RyR2 in HEK 293 cells stimulated with caffeine, but
agrees with studies by Tong et al., who did not detect endogenous RyR
expression in caffeine-stimulated HEK 293 cells
(Querfurth et al., 1998;
Tong et al., 1999
). HEK 293
cells expressing RyR1 or RyR3 recombinant proteins were stimulated with
increasing concentrations of caffeine (Fig.
4). RyR3-expressing cells were found to respond to lower
concentrations of caffeine than RyR1. Threshold values range between
0.125-0.250 mM for RyR1 (n=63) and 0.0625-0.1250 mM for RyR3
(n=58). In this study, we observed that RyR1 and RyR3 in HEK 293
cells display a different sensitivity to caffeine stimulation, with RyR3 being
more sensible to lower caffeine concentrations than RyR1, as noted previously
in myogenic cells (Fessenden et al.,
2000
). No systematic difference in the basal calcium levels
between the control and transfected cells was observed (data not shown).
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RyR3-expressing cells display spontaneous Ca2+ release
events
Confocal microscopy of Fluo-3-loaded cells indicated that the
RyR3-expressing cells displayed spontaneous subcellular
Ca2+-release events, similar to the Ca2+ sparks and
Ca2+ puffs previously described in various cell types
(Bootman et al., 1997a). In the
majority of the cells (>90%, n=300), the spontaneous localized
Ca2+ signals arose repetitively from a single site
(Fig. 5B), more frequently
occurring in a peripheral region of the cell, with the rest of the cell being
quiescent. No subcellular Ca2+ signals were recorded from control
cells (Fig. 5C). Similar
spontaneous activity was observed in five different RyR3-expressing clones,
which expressed different amounts of RyR3 protein. Irrespective of the level
of RyR3 expression, there was usually only a single spontaneously active site
within the cells. Why such spontaneous events are restricted to 1 or 2 points
in the endoplasmic reticulum, while RyR3 channels are widely distributed
within the endoplasmic reticulum is not clear. It is reasonable to envision
that occurrence of spontaneous Ca2+ events in specific sites could
be due to the presence of yet unidentified modulatory proteins at these
points. Although RyR1-expressing and RyR3 cells responded similarly to
caffeine stimulation, RyR1 cells did not display any spontaneous events either
when unstimulated (Fig. 5A) or
during applications of low (0.25-1.00 mM) caffeine concentrations (not shown).
The higher caffeine sensitivity, the capability of generating spontaneous
Ca2+ events in RyR3-expressing cells, as well as the
characteristics and frequency of these spontaneous events were not dependent
on the RyR level of expression, as they could be observed in cells with a RyR3
content as low as 41 fmol/mg of protein but not in cells with a RyR1 content
as high as 136.5 fmol/mg of protein.
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Characterisation of spontaneous Ca2+ events in
RyR3-expressing cells
The characteristics of the spontaneous and localized Ca2+
release events differed significantly among cells. Some sites produced signals
that peaked after a couple of hundred milliseconds and had a total duration of
up to a second (Fig. 6Aa). In
other cells, the Ca2+ signals were more reminiscent of the
Ca2+ sparks in cardiac muscle, with a rapid rising phase (time to
peak 50 mseconds) and a recovery time of 100-200 mseconds
(Fig. 6Ba). In addition to
differences in the temporal properties of the localized Ca2+
signals, the spatial spread of the signals was extremely variable, as shown by
the line-scan plots in Fig.
6Ab,Bb. To determine whether the spontaneous Ca2+
signals arose from the activation of RyRs, the cells were superfused with
ryanodine at a concentration of 100 µM, which has been shown to inhibit RyR
opening. Superfusion of cells with ryanodine for 5 minutes completely blocked
all spontaneous Ca2+-release events
(Fig. 6C). Application of
caffeine (40 mM) following inhibition of RyRs with ryanodine evoked only a
slight quench of the Fluo 3 fluorescence, with no obvious Ca2+
release (not shown).
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Discussion |
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Discrete localised increments of intracellular Ca2+, named
Ca2+ sparks, arising from co-operative opening of groups of few
RyRs, have been reported in cardiac
(Cannell and Soeller, 1999),
skeletal (Tsugorka et al.,
1995
; Klein et al.,
1996
) and smooth muscles
(Jaggar et al., 2000
;
Mironneau et al., 2001
;
Coussin et al., 2000
), and
neurons (Koizumi et al.,
1999
). Studies on embryonic skeletal muscle cells derived from
RyR1- or RyR3-knockout mice have revealed that both RyR isoforms have the
ability to produce, independently from each other, Ca2+ sparks with
nearly identical properties, although different from those observed in normal
muscles expressing both isoforms
(Shirokova et al., 1999
;
Conklin et al., 1999
;
Conklin et al., 2000
). In
contrast, transfection of RyR1 and RyR2 in non-muscle cells did not result in
the generation of Ca2+ sparks, from which it was concluded that
RyRs by themselves may not be sufficient to support generation of localized
Ca2+ release events and that a muscle-specific environment may be
required for the organisation of active Ca2+ release units and
generation of Ca2+ sparks (Bhat
et al., 1997
; Bhat et al.,
1999
). Accordingly, expression of heterologous RyR1 or RyR3 has
been shown to result in Ca2+ sparks in 1B5 muscle cells, which do
not express endogenous RyRs (Fessenden et
al., 2000
; Ward et al.,
2000
; Protasi et al.,
2000
). The ability of both RyR1 and RyR3 to generate
Ca2+-release activity in 1B5 skeletal muscle cells has been
confirmed recently (Ward et al.,
2001
). Comparison of the release events mediated by RyR1 or RyR3
channels in 1B5 cells led to the conclusion that, although both isoforms can
support spontaneous release events, the spatio-temporal properties and
frequency of events mediated by the two RyR isoforms differ.
In contrast to the above conclusions, results presented here clearly
indicate that spontaneous Ca2+-release events can be supported by
RyR3 channels in non-muscle cells, indicating that RyR3 channels themselves
are sufficient to support generation of localised Ca2+-release
activity. In contrast with the results obtained with HEK 293 cells expressing
RyR3, HEK 293 cells transfected with RyR1 did not display any spontaneous
Ca2+-release events either when unstimulated or following
application of low caffeine concentrations. The observation that RyR1 and
RyR3, when expressed under equivalent cellular conditions display a different
behaviour in terms of spontaneous activity suggests that isoform-specific
functional properties of RyR3 channels may be important for generation of
specific intracellular Ca2+ signals. Regulatory properties of RyR
channels have been extensively studied
(Meissner, 1994). Studies of
RyR3 channel properties have revealed that they can participate in
Ca2+-induced Ca2+-release processes, which may
facilitate the generation of Ca2+ sparks
(Murayama and Ogawa, 1996
;
Murayama and Ogawa, 1997
;
Murayama and Ogawa, 2001
;
DiJulio et al., 1997
;
Sonnleitner et al., 1998
;
Sorrentino and Reggiani,
1999
).
A second interesting observation is the finding that, in RyR3-expressing
cells, spontaneously generated Ca2+-release events were restricted
to one or, at most, two sites. Localised Ca2+ release events are
expected to result from the activation of a variable number of intracellular
Ca2+-release channels (Thorn et
al., 1993; Stricker,
1999
; Marchant and Parker,
2001
; Bootman et al.,
1997b
; Koizumi et al.,
1999
). The mechanism leading to the formation of these sites is
not clear, although clustering or organisation of channels in functional
Ca2+-release units is obviously required. In frog sympathetic
neurons Ca2+-release events have been found to arise in the cell
periphery, a region with high density of RyRs and SERCA pumps; this suggests
the presence of a functional specialisation of the endoplasmic reticulum that
may promote generation of localised Ca2+ release events
(McDonough et al., 2000
). In
transfected HEK 293 cells, RyR1 and RyR3 channels seemed to be uniformly
distributed throughout the endoplasmic reticulum, similarly to what has been
observed for other endoplasmic reticulum proteins. It cannot be excluded,
however, that either RyR channels or proteins of the endoplasmic reticulum
involved in Ca2+ signalling may organise at a level not detectable
by the techniques used.
In conclusion, we have found that, in HEK 293 cells, RyR1 and RyR3 channels are homogeneously distributed in the endoplasmic reticulum. These channels, when stimulated with caffeine, are able to release Ca2+ with a global response involving the entire endoplasmic reticulum. In contrast, expression of recombinant RyR3, but not RyR1, channels results in the appearance of Ca2+ sparks. These data provide direct evidence that differential expression of RyR isoforms may be important for generating specific patterns of Ca2+ signalling. In spite of initial attempts, we still have no evidence of what determines the formation of discrete Ca2+-release events in specific domains of the endoplasmic reticulum of HEK 293 cells transfected with RyR3 channels. More work is in progress to address this important point.
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Acknowledgments |
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