From the Laboratoire de Signalisation et Interactions Cellulaires, CNRS UMR 5017, Université de Bordeaux II, 146 rue Léo Saignat, Bordeaux Cedex 33076, France and the § Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37235
Received for publication, July 7, 2000, and in revised form, December 11, 2000
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ABSTRACT |
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Using an antisense strategy, we have previously
shown that in vascular myocytes, subtypes 1 and 2 of ryanodine
receptors (RYRs) are required for Ca2+ release during
Ca2+ sparks and global Ca2+ responses, evoked
by activation of voltage-gated Ca2+ channels, whereas RYR
subtype 3 (RYR3) has no contribution. Here, we investigated the effects
of increased Ca2+ loading of the sarcoplasmic reticulum
(SR) on the RYR-mediated Ca2+ responses and the role of the
RYR3 by injecting antisense oligonucleotides targeting the RYR
subtypes. RYR3 expression was demonstrated by immunodetection in both
freshly dissociated and cultured rat portal vein myocytes. Confocal
Ca2+ measurements revealed that the number of cells showing
spontaneous Ca2+ sparks was strongly increased by
superfusing the vascular myocytes in 10 mM
Ca2+-containing solution. These Ca2+ sparks
were blocked after inhibition of RYR1 or RYR2 by treatment with
antisense oligolucleotides but not after inhibition of RYR3. In
contrast, inhibition of RYR3 reduced the global Ca2+
responses induced by caffeine and phenylephrine, indicating that RYR3
participated together with RYR1 and RYR2 to these Ca2+
responses in Ca2+-overloaded myocytes. Ca2+
transients evoked by photolysis of caged Ca2+ with
increasing flash intensities were also reduced after inhibition of RYR3
and revealed that the [Ca2+]i sensitivity of RYR3
would be similar to that of RYR1 and RYR2. Our results show that, under
conditions of increased SR Ca2+ loading, the RYR3 becomes
activable by caffeine and local increases in
[Ca2+]i.
Since the description of a Ca2+-induced
Ca2+ release mechanism in skeletal muscle (1), the function
of ryanodine receptor channels (RYRs)1 have been widely
studied in both skeletal and cardiac muscles (2-7) and more recently
in smooth muscle (8, 9). After the cloning and sequencing of three
genes encoding different RYR subtypes, the localization and role of
each RYR subtype in Ca2+ signaling have begun to be
studied. Of the three RYRs, RYR subtype 3 (RYR3) is the most widely
expressed (10), whereas RYR1 and RYR2 are mainly found in skeletal and
cardiac muscles, respectively (11, 12). However, in arterial and venous
smooth muscles, the three RYR subtypes have been identified (13, 14),
but their role in Ca2+ release is still unclear.
The regulation of the different RYR subtypes has been extensively
studied using single channel recordings in lipid bilayers. The single
channel properties of RYR subtypes are rather similar, i.e.
they form a large conductance channel permeable to monovalent and
divalent cations, which can be activated by ATP, caffeine, and
submicromolar concentrations of Ca2+; inhibited by
Mg2+, ruthenium red, and millimolar concentrations of
Ca2+; and modulated by ryanodine (15-17). However,
differences in responses to cyclic ADP-ribose and caffeine as well as
in sensitivities to Ca2+ activation and Ca2+
inactivation have been reported between RYR1 and RYR3 and between RYR3s
cloned from skeletal and smooth muscles (17-20). It has been proposed
that these differences may result from the expression of different
splice variants of RYR3 (21).
The physiological contribution of the different RYR subtypes to
Ca2+ signaling has been addressed first by using either
RYR1 and RYR2 knockout mice (12, 22) or RYR3 knockout mice (23-25). In
RYR1-null myotubes in culture, the Ca2+ release from the
sarcoplasmic reticulum (SR) in response to increases in cytosolic
Ca2+ concentration ([Ca2+]i) or
caffeine is strongly reduced, but a similar decrease in caffeine
sensitivity is also observed in RYR3-null neonatal myocytes, suggesting
a possible co-contribution of each RYR subtype to Ca2+
signaling, at least at some stages of myogenesis. Accordingly, it has
been recently proposed that, in embryonic skeletal muscle, both RYR1
and RYR3 may co-contribute to Ca2+ release during
Ca2+ sparks (25). In addition, Ca2+ sparks
produced independently in RYR1- or RYR3-null cells reveal similar
spatio-temporal parameters (26). Another approach using antisense
oligonucleotides, that specifically targeted each one of the RYR
subtypes, has been used to determine which RYR subtypes are responsible
for Ca2+ sparks and global Ca2+ responses in
smooth muscle cells (14). It appears that both RYR1 and RYR2 are
required for Ca2+ release during Ca2+ sparks
and Ca2+ waves induced by activation of L-type
Ca2+ currents and that RYR3 does not contribute to
these Ca2+ signals.
In smooth muscle cells, Ca2+ sparks are observed
spontaneously or in response to Ca2+ influx through L-type
Ca2+ channels (8, 9, 27, 28), and their localization
corresponds to coupling areas between the plasma membrane and the SR
(28, 29). In rat portal vein myocytes, spatial and temporal recruitment of Ca2+ sparks results in propagating Ca2+
waves, which trigger cell contraction (30).
In the present study, we investigated the effects of elevating the
extracellular Ca2+ concentration
([Ca2+]o) on both Ca2+ sparks and
global Ca2+ responses induced by Ca2+,
caffeine, and phenylephrine. Under conditions of increased SR Ca2+ loading, we provide the first evidence that the RYR3,
which becomes activable by caffeine and localized increases in
[Ca2+]i, is responsible for the increased global
Ca2+ responses. We also found that spontaneous
Ca2+ sparks are highly abundant but remain dependent on
activation of only RYR1 and RYR2.
Cell Preparation--
Rats (160-180 g) were killed by cervical
dislocation. The portal vein was cut into several pieces and incubated
for 10 min in low Ca2+ (40 µM) physiological
solution, and then 0.8 mg/ml collagenase (EC 3.4.24.3), 0.20 mg/ml
Pronase E (EC 3.4.24.31), and 1 mg/ml bovine serum albumin were added
at 37 °C for 20 min. After this time, the solution was removed, and
pieces of portal vein were incubated again in a fresh enzyme solution
at 37 °C for 20 min. Tissues were placed in a enzyme-free solution
and triturated using fire-polished Pasteur pipette to release cells.
Cells were seeded at a density of 103 cells/mm2
on glass slides imprinted with squares for localization of injected cells. Cells were maintained in short term primary culture in medium
M199 containing 2% fetal calf serum, 2 mM glutamine, 1 mM pyruvate, 20 units/ml penicillin, and 20 µg/ml
streptomycin; they were kept in an incubator gassed with 95% air and
5% CO2 at 37 °C. The myocytes were cultured in this
medium for 4 days. The normal physiological solution contained 130 mM NaCl, 5.6 mM KCl, 1 mM
MgCl2, 1.7 mM CaCl2, 11 mM glucose, and 10 mM HEPES (pH 7.4, with NaOH).
Microinjection of Oligonucleotides--
Phosphorothioate
antisense oligonucleotides (denoted with the prefix "as") used in
the present study were designed on the known cloned RYR sequences
deposited in the GenBankTM sequence data base with Lasergene software
(DNASTAR, Madison, WI). Sequences of all three RYR cDNAs were
aligned with each other, and specific antisense oligonucleotide
sequences were chosen in region of the cDNA of interest, completely
different from the sequences of the two other RYR subtypes. Then
antisense and scrambled sequences displaying putative binding to any
other mammalian sequences deposited in GenBankTM were discarded.
Oligonucleotides were injected into the nuclei of myocytes by a manual
injection system (Eppendorf, Hamburg, Germany). Intranuclear
oligonucleotide injection with Femtotips II (Eppendorf) was performed
as previously described (14). The myocytes were then cultured for 3-4
days in culture medium, and the glass slides were transferred into the
perfusion chamber for physiological experiments. The sequences of
as1RYR1 and as2RYR1 are AGCGTGTGCAGCAGGCTCA and GCAATCCGCTCCCGCCCA,
corresponding to nucleotides 325-343 and 584-601, respectively, of
RYR1 cDNA deposited in GenBankTM (accession number
X83932); those of as1RYR2 and as2RYR2 are GTGTCCTCACAGAAGTT and
TGAAATCTAGTGCAGCCT, corresponding to nucleotides 137-153 and
1587-1604, respectively, of RYR2 cDNA (accession number X83933);
and those of as1RYR3 and as2RYR3 are AAGTCAAGGGCATTTTTG and
ACTTAGCCATGACACCAG, corresponding to nucleotides 502-519 and 557-574,
respectively, of RYR3 cDNA (accession number X83934). In some
control experiments, myocytes were injected with the following scrambled oligonucleotides: CACGCCTACGCACCTCCG, corresponding to a
scrambled sequence of as2RYR1 (nucleotides 584-601 of RYR1 cDNA);
AGTCGTACATGACTCGTA, corresponding to a scrambled sequence of as2RYR2
(nucleotides 1587-1604 of RYR2 cDNA); and CAGCACTATCAGTACGAC, corresponding to a scrambled sequence of as2RYR3 (nucleotides 557-574
of RYR3 cDNA).
Cytosolic Ca2+ Measurements--
In most
experiments, cells were loaded by incubation in physiological solution
containing 4 µM fluo 3-acetoxymethylester (fluo 3-AM) for
1 h at room temperature. These cells were washed and allowed to
cleave the dye to the active fluo 3 compound for at least 30 min.
Images were acquired using the line scan mode of a confocal Bio-Rad
MRC1000 microscope connected to a Nikon Diaphot microscope. Excitation
light was delivered by a 25-milliwatt argon ion laser (Ion Laser
Technology, Salt Lake City, UT) through a Nikon Plan Apo × 60, 1.4 NA objective lens. Fluo 3 was excited at 488 nm, and emitted
fluorescence was filtered and measured at 540 ± 30 nm. At the
setting used to detect fluo 3 fluorescence, the resolution of the
microscope was near 0.4 × 0.4 × 1.5 µm (x, y, and z axis). Images were acquired in the line
scan mode at a rate of 6 ms/scan. Scanned lines were plotted
vertically, and each line was added to the right of the preceding line
to form the line scan image. In these images, time increased from the left to the right, and position along the scanned line was given by
vertical displacement. Fluorescence signals are expressed as pixel per
pixel fluorescence ratios (F/Fo), where
F is the fluorescence during a response and
Fo is the rest level fluorescence of the same pixel.
Image processing and analysis were performed by using COMOS, TCSM, and
MPL 1000 software (Bio-Rad).
In other experiments, cells were loaded by incubation in physiological
solution containing 1 µM indo 1-AM for 30 min.
[Ca2+]i measurements were estimated from the
405-/480-nm fluorescence ratio, as previously reported (31). The
minimum and maximum fluorescence (Rmin and
Rmax, respectively) values were determined in vivo, in the absence of Ca2+ and at
saturating Ca2+, in cells superfused in 1.7 and 10 mM [Ca2+]o.
Caffeine and phenylephrine were applied by pressure ejection from a
glass pipette for the period indicated on the records. All experiments
were carried out at 26 ± 1 °C.
Flash Photolysis--
Caged Ca2+,
1-(4,5-dimethoxy-2-nitrophenyl)-EDTA, tetra(acetoxymethylester)
(DMNP-EDTA, AM) at 15 µM, was added to the bathing solution and maintained in the presence of cells for 1 h in an incubator at 37 °C. Photolysis was produced by a 1-ms pulse from a
xenon flash lamp (Hi-Tech Scientific, Salisbury, United Kingdom) focused to a ~2-mm diameter spot around the cell. Light was band pass-filtered with a UG11 glass between 300 and 350 mm. Flash intensity
could be adjusted by varying the capacitor-charging voltage between 0 and 380 V, which corresponded to a change in the energy input into the
flash lamp from 0 to 240 J. On flash photolysis, Ca2+ was
released within 2 ms, and the small percentage of conversion of the
caged compound (~10%) allowed us to apply repetitive pulses without
altering the Ca2+ responses and the reserve of caged
Ca2+.
RYR Labeling--
Freshly dissociated and cultured myocytes (3 days after injection) were immunostained as previously described (30).
Briefly, myocytes were incubated in the presence of anti-RYR3-specific antibody (20) (at 1:100 dilution) for 20 h at 4 °C and with the
secondary antibody (donkey anti-rabbit IgG conjugated to fluorescein isothiocyanate, diluted at 1:200) for 3 h at 20 °C. Thereafter, cells were mounted in Vectashield. Images of the stained cells were
obtained with the Bio-Rad confocal microscope. Control cells and
injected cells on the same glass slide were compared with each other by
keeping acquisition parameters constant (gray values, exposure time,
aperture). Fluorescent labeling was estimated by gray level analysis
using MPL software and expressed in arbitrary units of fluorescence.
Chemicals and Drugs--
Collagenase was obtained from
Worthington. Fluo 3-AM and DMNP-EDTA, AM were from Molecular
Probes (Leiden, The Netherlands). Caffeine was from Merck. Indo 1-AM,
ryanodine, and cyclopiazonic acid were from Calbiochem. Medium M199 was
from ICN (Costa Mesa, CA). Fetal calf serum was from Bio Media
(Boussens, France). Streptomycin, penicillin, glutamine, and pyruvate
were from Life Technologies, Inc. All primers and phosphorothioate
antisense oligonucleotides were synthesized and purchased from
Eurogentec (Seraing, Belgium). All other chemicals were from Sigma. The
rabbit anti-RYR3-specific antibody was directed against the deduced
amino acid sequence, 4326-4336 (11 amino acids), of rabbit RYR3
(20).
Data Analysis--
Data are expressed as means ± S.E.;
n represents the number of tested cells. Significance was
tested by means of Student's test. p values < 0.05 were considered as significant.
Effects of High Ca2+-containing Solution on the
Caffeine-induced Ca2+ Responses in Vascular
Myocytes--
The effects of increasing the external Ca2+
concentration ([Ca2+]o) in rat portal vein
myocytes were studied in a series of experiments, in which caffeine (10 mM) was applied through a micropipette positioned near the
surface of the cells and the resulting [Ca2+]i
changes were measured either in the entire cytosol (indo 1 experiments)
or in a single line repeatedly scanned through the confocal cell
section (fluo 3 experiments). In indo 1-loaded cells, the peak
fluorescence (
To assess the role of the SR Ca2+ loading in the generation
of large and fast Ca2+ responses to caffeine, the effects
of 10 µM cyclopiazonic acid were first investigated on
the caffeine-induced Ca2+ responses. Inhibition of the
Ca2+ uptake capacity of the intracellular store by
cyclopiazonic acid resulted in a small elevation of the basal
[Ca2+]i and the suppression of the
caffeine-induced Ca2+ response in the continuous presence
of cyclopiazonic acid for 5 min (n = 6). In a second
set of experiments, caffeine (10 mM) was applied in
Ca2+-free, 0.5 mM EGTA-containing solution for
10 s (a time sufficient to remove voltage-dependent
Ca2+ current) on myocytes superfused either in 1.7 mM [Ca2+]o or 10 mM
[Ca2+]o. After 10 s in Ca2+-free
solution, the amplitude of the caffeine-induced Ca2+
responses (measured with indo 1) was 0.55 ± 0.03 ( Antisense Oligonucleotide Strategy--
As recently published
(14), we designed antisense oligonucleotides specifically targeting
each RYR subtype mRNA. For each RYR subtype, two antisense
sequences were chosen, one targeting the region of the mRNA
amplified in PCR experiments (named as2RYR) and the other one (named
as1RYR) designed to hybridize the mRNA outside the amplified
fragment but close to the start codon. The time course of antisense
oligonucleotide efficiency was determined by checking the ability of a
mixture of as1RYR1 + as1RYR2 + as1RYR3 (10 µM each) to
inhibit the Ca2+ waves induced by 10 mM
caffeine in isolated myocytes superfused in 10 mM
[Ca2+]o for 1 h. The Ca2+
responses were strongly inhibited 3 days after nuclear injection of the
antisense oligonucleotides (83 ± 5%, n = 30);
recovery began the fourth day with an inhibition of 48 ± 5%
(n = 25). Nonspecific effects of antisense
oligonucleotides were detected only at concentrations higher
than 50 µM (for example, inhibition of RYR2 protein
expression by 50 µM anti-G
Immunodetection of RYR3 with an anti-RYR3-specific antibody (20)
revealed a homogeneous distribution of fluorescence in cell sections
from freshly isolated and cultured myocytes (Fig. 2, A and B). In
cells injected with as1RYR3, the immunostaining was very weak (Fig.
2C), whereas it was not significantly changed in cells
injected with as1RYR1 + 2 (Fig. 2, E and F).
These results indicate that RYR3 is expressed in rat portal vein
myocytes and can be selectively inhibited by asRYR3 without
variation in the expression of the other RYR subtypes; they are in good
agreement with previous data using BODIPY®-labeled ryanodine staining,
which showed that inhibition of each one of the three RYR subtypes
decreased by approximately one-third the specific fluorescence
(14).
RYR Subtypes Involved in Ca2+ Sparks under Increased SR
Ca2+ Loading--
We have previously reported that, in 1.7 mM [Ca2+]o, both RYR1 and RYR2 are
required for Ca2+ release during Ca2+ sparks
evoked by activation of voltage-gated Ca2+ channels (14).
Since elevation of luminal [Ca2+] has been suggested to
increase the activity of RYRs (32), we studied the parameters of
spontaneous Ca2+ sparks in Ca2+-overloaded
cells. In control cells, superfused in 1.7 mM
[Ca2+]o, spontaneous Ca2+ sparks were
rarely detected, in less than 25% of cells tested (7/32 cells), and
the number of initiation sites per line scan image was 1.1 ± 0.1 (n = 7). In 10 mM
[Ca2+]o, spontaneous Ca2+ sparks were
detected in about 80% of cells tested (116/140 cells; Fig.
3A), and the number of
initiation sites per line scan image was 2.17 ± 0.21 (n = 44, Fig. 3C). In contrast, the
spatio-temporal parameters of Ca2+ sparks were not
significantly different in 1.7 mM
[Ca2+]o and 10 mM
[Ca2+]o (Table
I).
When cells were injected with asRYR1, asRYR2, or asRYR1 + 2, the number
of cells with spontaneous Ca2+ sparks was strongly
decreased (Fig. 3A). In contrast, the number of cells with
spontaneous Ca2+ sparks and the number of initiation sites
per line scan image were not significantly affected in cells injected
with asRYR3 (Fig. 3, B and C). These results
suggest that under both normal (14) and increased SR Ca2+
content conditions, Ca2+ sparks are due to activation of
both RYR1 and RYR2 and that RYR3 does not contribute to triggering
Ca2+ sparks. In addition, the spatio-temporal parameters of
Ca2+ sparks in 10 mM
[Ca2+]o were not significantly different in
noninjected cells and in cells injected with asRYR3 (Table I). Taken
together, these results suggest that increased SR Ca2+
loading potentiates the activity of Ca2+ release units
formed by RYR1 and RYR2, leading to an increase in Ca2+
spark frequency without alterations of the spatio-temporal parameters.
RYR Subtypes Involved in Caffeine-induced Ca2+
Responses under Increased SR Ca2+ Loading--
We have
previously shown that in 1.7 mM
[Ca2+]o, caffeine triggers Ca2+ waves
by activating both RYR1 and RYR2 (14). Consequently, inhibition of RYR1
or RYR2 by treatment with antisense oligonucleotides partly inhibited
the caffeine-induced Ca2+ responses, whereas inhibition of
RYR3 was ineffective (14). Using the same anti-RYR antisense
oligonucleotides, we determined the role of each RYR subtype in the
generation of the caffeine-induced Ca2+ responses under
conditions of increased SR Ca2+ loading. Cells injected
with asRYR1 or asRYR2 evoked Ca2+ responses to 10 mM caffeine that were similar to those elicited in control
cells superfused in 10 mM [Ca2+]o
(Fig. 4A). However, injection
of both as1RYR1 + as1RYR2 and injection of asRYR3 alone significantly
decreased the caffeine-induced Ca2+ responses by 55 and
35%, respectively (Fig. 4, A and B). Maximal inhibition (85%) was obtained in cells injected with a mixture of
as1RYR1 + 2 + 3 (Fig. 4A). Scrambled RYR3 antisense
oligonucleotides did not affect significantly the Ca2+
responses evoked by caffeine (Fig. 4B). It is noteworthy
that the upstroke velocities of the Ca2+ responses in
as1RYR1- and as1RYR2-injected cells were not significantly different
from that measured in control cells, whereas that in as1RYR3-injected
cells was decreased by 65% (Fig. 4C). Although injection of
as1RYR1 + 2 decreased the upstroke velocity of the caffeine-induced
Ca2+ response, this inhibition was significantly less than
that obtained in as1RYR3-injected cells (Fig. 4C). These
results indicate that the RYR3 becomes activable by caffeine in cells
preloaded with high [Ca2+]o. Since inhibition of
RYR1 or RYR2 can be compensated by activation of RYR3, these results
suggest that the biophysical properties of the RYR3 may be different
from those of RYR1 and RYR2.
RYR Subtypes Involved in [Ca2+]i Jump- and
Phenylephrine-induced Ca2+ Responses--
To determine the
effects of a direct increase in [Ca2+]i on
activation of the RYR subtypes, flash photolysis of DMNP-EDTA was used
to instantaneously elevate the [Ca2+]i, as
previously reported (28). As illustrated in Figs.
5 (A and B) and
6A, both amplitude and full time at half-maximal amplitude
(FTHM) of the Ca2+ responses evoked by a flash
pulse of 66 J were increased significantly in 10 mM
Ca2+-pretreated cells when compared with control cells
superfused in 1.7 mM [Ca2+]o. The
full time at half-maximal amplitude increased from 0.41 ± 0.02 s (n = 21) in 1.7 mM
[Ca2+]o to 0.56 ± 0.03 s
(n = 27) in 10 mM
[Ca2+]o. The increase in
[Ca2+]i due to Ca2+ release from the
caged molecule in response to a 66-J flash pulse was estimated in cells
pretreated with 100 µM ryanodine for 20 min to block all
of the RYRs (Fig. 6A). In
cells injected with as2RYR3, the amplitude of the Ca2+
responses evoked by flash photolysis of caged Ca2+ in 10 mM [Ca2+]o was significantly
decreased (Figs. 5C and 6B) and became similar to
that obtained in control cells superfused in 1.7 mM
[Ca2+]o (Figs. 5A and 6A).
In contrast, inhibition of RYR1 or RYR2 by treatment with antisense
oligonucleotides had no significant effects on the Ca2+
responses evoked by flash photolysis of caged Ca2+ in 10 mM [Ca2+]o (Fig. 6B).
However, injection of as2RYR1 + 2 significantly attenuated the
Ca2+-induced Ca2+ responses (Fig.
6B). To evaluate the Ca2+ sensitivity of the RYR
subtypes to [Ca2+]i, the amplitude of the
Ca2+ transients obtained from the entire line scan images
was plotted as a function of flash intensity in control cells and after
inhibition of the RYR subtypes in 10 mM
[Ca2+]o. As expected, the
Ca2+-induced increase in [Ca2+]i was
reduced by treatment with as2RYR3 and as2RYR1 + 2 at all of the flash
intensities tested (Fig. 7A).
Maximal inhibition was obtained in cells pretreated with 100 µM ryanodine for 20 min (Fig. 7A). It was
noted that the curve obtained in 1.7 mM
[Ca2+]o was similar to that obtained in 10 mM [Ca2+]o after inhibition of RYR3s
(Fig. 7A). The Ca2+ sensitivity of RYR subtypes
was examined by plotting the ratio between the peak Ca2+
transients and the maximal Ca2+ transient, at different
flash intensities in cells superfused in 10 mM
[Ca2+]o, before and after inhibition of RYR1 + 2 or RYR3 alone (Fig. 7B). The curves were practically
superimposed, suggesting that the Ca2+ sensitivities of the
three RYR subtypes to [Ca2+]i could be similar.
These results show that in Ca2+-overloaded cells, the RYR3
is also activable by local increases in
[Ca2+]i.
We have reported previously that norepinephrine may activate RYRs
following Ca2+ release via inositol
1,4,5-trisphosphate-gated Ca2+ channels (30). In
Ca2+-overloaded cells, both amplitude and upstroke velocity
of the Ca2+ waves induced by 10 µM
phenylephrine ( Growing evidence suggests that the activity of RYRs of the SR can
be influenced by luminal Ca2+ (32-34), but the
contribution of the different RYR subtypes in increased
Ca2+ release has never been investigated. Based on the
antisense oligonucleotide strategy, the present study shows that, in
rat portal vein myocytes, the RYR3 becomes activable under conditions
of increased SR Ca2+ loading and is responsible for the
increase in Ca2+ release during caffeine- and
neuromediator-induced global Ca2+ responses. We confirm the
contribution of both RYR1 and RYR2 but not of RYR3 to Ca2+
sparks in Ca2+-overloaded myocytes. Immunologic detection
of RYR3s revealed that these receptors were present in freshly
dissociated and cultured portal vein myocytes. Since the cellular
distribution of RYR3 was homogeneous in the cell sections, this
observation supports the idea that RYR3s do not constitute
Ca2+ release units.
Elevation of extracellular [Ca2+] created the conditions
for an increased SR Ca2+ loading, as previously reported in
cardiac myocytes (35, 36), except that a pretreatment of 1 h in 10 mM [Ca2+]o was needed for vascular
myocytes to reach a steady state. We found that inhibition of
Ca2+ accumulation into the SR by cyclopiazonic acid
completely suppressed the caffeine-induced Ca2+ responses,
whereas removal of external Ca2+ for 10 s had no
effect on the increased caffeine-induced Ca2+ responses in
10 mM [Ca2+]o, indicating that they
were strictly dependent on Ca2+ release from the SR.
Under the conditions of increased SR Ca2+ loading, the
frequency of spontaneous Ca2+ sparks was notably increased.
The number of vascular myocytes showing spontaneous Ca2+
sparks was enhanced from less than 25% of cells tested to more than
80%, and the number of initiation sites per line scan image was higher
than 2. A similar increase in Ca2+ spark frequency has been
reported after a SR Ca2+ overloading in ventricular
myocytes (37). Other experiments performed in stomach smooth muscle
cells have shown that the Ca2+ spark frequency is dependent
on the luminal Ca2+ concentration (38), but the RYR
subtypes involved in this modulation have not been identified.
Inhibition of either RYR1 or RYR2 by treatment with antisense
oligonucleotides strongly reduced the number of vascular myocytes with
spontaneous Ca2+ sparks, whereas inhibition of RYR3 was
ineffective. Interestingly, inhibition of both RYR1 and RYR2 was not
additive compared with inhibition of one RYR subtype, in accordance
with our previous data showing that both RYR1 and RYR2 are required for
activation of Ca2+ sparks under conditions of normal
Ca2+ loading (14). The fact that spots of fluorescence were
not detected in cells stained with an anti-RYR3-specific antibody (20)
after inhibition of both RYR1 and RYR2 by treatment with antisense
oligonucleotides supports the idea that RYR3s do not form clustered
units and, therefore, could not give rise to Ca2+ sparks.
This is in contrast with recent data showing that Ca2+
sparks with nearly identical properties were obtained in both RYR1- and
RYR3-null embryonic skeletal cells (26). Moreover, the characteristics
of Ca2+ sparks (i.e. the mean amplitude,
time-to-peak, full time at half-maximal amplitude, and full width at
half-maximal amplitude) (Table I), measured in noninjected cells and in
cells injected with asRYR3, were not significantly different,
supporting the idea that only RYR1 and RYR2 are engaged in
Ca2+ spark activity in vascular myocytes. Several
possibilities can be proposed to explain how increased luminal
Ca2+ concentration regulates RYRs activity and enhances the
number of spontaneous Ca2+ sparks. First, increased luminal
[Ca2+] may exert an allosteric modulation of RYRs
involving a luminal regulatory site, as previously suggested in cardiac
myocytes (32). Second, it may increase the conductance as well as the
open probability of RYR1 and RYR2. Our observations that the
Ca2+ spark parameters were not modified in 10 mM [Ca2+]o and the data obtained from
single channel experiments showing that increased luminal
[Ca2+] enhanced the number of openings rather than their
duration (32) do not support this proposal. Third, it may directly
activate silent Ca2+ release units. This possibility would
be consistent with previous observations that increased luminal
[Ca2+] activated only RYRs affected by other modulators
such as ATP (33). At the present time, these three possibilities cannot be clearly distinguished.
The sensitivity of RYR subtypes to [Ca2+]i jumps
was evaluated by releasing Ca2+ by flash photolysis of
caged Ca2+. The absence of shift for the normalized curves
obtained in 10 mM [Ca2+]o, in control
cells and in asRYR1 + 2-injected or asRYR3-injected cells, suggests
that the three RYR subtypes could have a similar sensitivity to
activation by local increases in [Ca2+]i. We have
previously estimated that the threshold Ca2+ level for
activation of Ca2+ sparks was ~95 nM, a value
that is not significantly different from the threshold for activation
of the purified native RYR1 from rabbit skeletal muscle and of the RYR3
from rabbit uterus in HEK293 cells (18). However, other authors found a
less sensitivity for Ca2+ ions of the RYR3 from skeletal
muscle (17, 19, 20). This difference might be related to
tissue-specific alternative splicing of RYR3, since rat portal vein
myocytes expressed predominantly RYR3-I
mRNA2 in contrast to
mouse skeletal muscle, which expressed only RYR3-II mRNA (21).
Although activation of RYR3s does not give rise to Ca2+
sparks in vascular myocytes, our results obtained with flash photolysis
of caged Ca2+ and phenylephrine applications indicate that
local increases in [Ca2+]i induced experimentally
or in response to activation of inositol 1,4,5-trisphosphate-gated
channels are sufficiently high to activate RYR3s under conditions of
increased SR Ca2+ loading. Therefore, our results support
the idea that RYR3 needs a previous increase in luminal
[Ca2+] to be activable by local increases in
[Ca2+]i. Furthermore, when cells were injected
with either asRYR1 or asRYR2, both caffeine- and
Ca2+-induced Ca2+ responses were not
significantly affected, suggesting that activation of RYR3s might
compensate for the inhibition of RYR1s or RYR2s. This observation is in
good agreement with the fact that the single channel properties of the
RYR3 differ from those of RYR1. For example, the maximal open
probability of the RYR3 activated by Ca2+ alone is close to
unity, whereas that of the RYR1 is about 0.2-0.5, and the RYR3 is
about 10 times less sensitive to inactivation by high Ca2+
concentrations than the RYR1 (17, 18). Recent data have confirmed that
the recombinant RYR3, expressed in HEK293 cells, is sensitive to
nanomolar [Ca2+] in the presence of 1 mM ATP
and does not present inactivation at high Ca2+
concentration (41). Our observations that the upstroke velocity of the
caffeine-induced Ca2+ response in asRYR1 + 2 is higher than
that obtained in asRYR3 is in accordance with the high open probability
of RYR3 compared with that of the other RYR subtypes.
Functional cells regulate their Ca2+ homeostasis, in part,
by Ca2+ uptake into the SR and Ca2+ efflux to
the extracellular space through specific Ca2+-ATPases,
suggesting that localized and transient Ca2+ overloads of
the SR may be of physiological relevance. Recently, potentiation of
excitation-contraction coupling in cardiac myocytes has been correlated
with an increase in free SR Ca2+ content (42). Since both
RYR1 and RYR2 once activated are precluded from rapid reactivation as a
result of RYR adaptation (40) or inactivation by Ca2+ ions
(39), it can be postulated that the RYR3, which has been reported to be
resistant to high Ca2+ concentrations (17, 18, 41), may
maintain Ca2+ release when the other RYR subtypes are closed.
In conclusion, these results show that, in vascular myocytes, RYR3 can
be activated by caffeine and local increases in
[Ca2+]i, under conditions of increased SR
Ca2+ loading.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ratio 405/480 nm) as well as the total fluorescence
measured during 10-s applications of caffeine (
ratio × 10 s) increased in a time-dependent manner and reached a steady-state value within 1 h in 10 mM
Ca2+-containing solution. In 1.7 mM
[Ca2+]o, the values for the peak and total
fluorescence were 0.69 ± 0.03 and 119.2 ± 7.6, respectively
(n = 46). In 10 mM
[Ca2+]o, these values increased to 0.98 ± 0.03 (n = 79) and 272.2 ± 26.1 (n = 46), respectively, indicating a 40% increase in caffeine response
amplitude and a 230% increase in SR Ca2+ release. In
contrast, the basal [Ca2+]i was not significantly
increased (from 55 ± 8 nM in 1.7 mM
[Ca2+]o to 64 ± 10 nM in 10 mM [Ca2+]o, n = 46).
In fluo 3-loaded cells (Fig. 1), the
amplitude of the caffeine-induced Ca2+ waves, measured from
a 2-µm region of the line scan image, also increased from 2.01 ± 0.10 (
F/Fo, n = 32)
in 1.7 mM [Ca2+]o to 2.66 ± 0.08 (n = 45) in 10 mM
[Ca2+]o, indicating a 30% increase in
Ca2+ response amplitude (Fig. 1B). Furthermore,
the upstroke velocity of the caffeine-induced Ca2+
response, corresponding to the initiation site of the response, was
enhanced from 8.57 ± 0.71 (
F/Fo·s
1,
n = 32) in 1.7 mM
[Ca2+]o to 33.57 ± 4.27 (n = 45) in 10 mM [Ca2+]o, indicating a
350% increase in Ca2+ release velocity (Fig.
1B). Taken together, these results suggest that the SR
Ca2+ content of vascular myocytes is increased by sustained
elevation in extracellular [Ca2+] and that both amplitude
and upstroke velocity of the Ca2+ responses can be used as
significant parameters to study the cellular mechanisms involved during
increased SR Ca2+ loading.
View larger version (28K):
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Fig. 1.
Effects of increased
[Ca2+]o on caffeine-induced
[Ca2+]i responses in rat portal vein
myocytes. A, line scan images of fluorescence changes
induced by 10 mM caffeine in 1.7 mM
Ca2+-containing solution (a) and 1 h after
increasing [Ca2+]o to 10 mM
(b). Traces show (from top to bottom)
caffeine application, line scan image, and averaged fluorescence
from 2-µm regions (indicated by the small
vertical bars) of the line scan image.
B, compiled data showing the effects of 10 mM
[Ca2+]o on both amplitude and upstroke velocity
of caffeine-induced Ca2+ responses. Caffeine was applied at
10 mM. Bars show means ± S.E. in 1.7 mM [Ca2+]o (open
bars) and 10 mM [Ca2+]o
(filled bars), with the number of cells tested
indicated in parentheses. Cells were obtained from five
different batches. , values significantly different from those
obtained in 1.7 mM [Ca2+]o. Myocytes
were loaded with fluo 3-AM.
ratio,
n = 26) in myocytes pretreated with 1.7 mM
[Ca2+]o and 0.79 ± 0.07 (n = 26) in myocytes pretreated with 10 mM
[Ca2+]o, indicating an increase in
Ca2+ response amplitude similar to that obtained in
Ca2+-containing solutions (about 40%). Taken together,
these results suggest that the increased accumulation of
Ca2+ in the SR is responsible for the large and fast
caffeine-induced Ca2+ responses under conditions of
increased [Ca2+]o.
o antisense oligonucleotide).
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Fig. 2.
Efficiency of RYR antisense
oligonucleotides. Typical labeling of RYR3 is obtained with an
anti-RYR3 specific antibody (20) in freshly isolated cells
(A), in cells cultured for 4 days in control conditions
(B), and after nuclear injection of as1RYR3 alone
(C) or a mixture of as1RYR1 + 2 (E).
D, scanning transmission image of the cell shown in
C. F, compiled data expressed in arbitrary units
(AU) of anti-RYR3 specific antibody fluorescence measured in
control and injected cells. Nonspecific fluorescence was estimated in
the presence of 2 µg/ml antigen peptide (hatched
bar). Open bars (noninjected control
cells) and filled bars (cells injected with
asRYRs) show means ± S.E., with the number of cells tested
indicated in parentheses. Cells were obtained from three
different batches. , values significantly different from those
obtained in noninjected cells. Cells were immunostained 3 days after
nuclear injection of antisense oligonucleotides. N,
nucleus.
View larger version (27K):
[in a new window]
Fig. 3.
Effects of RYR antisense oligonucleotides on
the spontaneous Ca2+ sparks in 10 mM
[Ca2+]o. A, percentage of cells
showing spontaneous Ca2+ sparks in 1.7 mM
[Ca2+]o and in 10 mM
[Ca2+]o either in noninjected cells
(open bars) or in cells injected with 10 µM asRYR1, 10 µM asRYR2, a mixture of
as1RYR1 + 2, or a mixture of as1RYR1 + 2 + 3 at 10 µM
each (filled bars). B, percentage of
cells with spontaneous Ca2+ sparks in 10 mM
[Ca2+]o, in noninjected cells (open
bars) and in cells injected with 10 µM as1RYR3
or as2RYR3 (filled bars). C, number of
initiation sites triggering spontaneous Ca2+ sparks per
line scan image in 10 mM [Ca2+]o, in
noninjected cells (open bars) and in cells
injected with 10 µM as1RYR3 or as2RYR3 (filled
bars). Bars show means ± S.E. in
noninjected control cells (open bars) or in cells
injected with asRYRs (filled bars), with the
number of cells tested indicated in parentheses. Cells were
obtained from five different batches. , values significantly
different from those obtained in noninjected cells in 10 mM
[Ca2+]o. Myocytes were loaded with fluo
3-AM.
Effects of RYR3 antisense oligonucleotides on the parameters of
Ca2+ sparks
View larger version (30K):
[in a new window]
Fig. 4.
Effects of RYR antisense oligonucleotides on
caffeine-induced Ca2+ responses in 10 mM
[Ca2+]o. Caffeine was applied at 10 mM. A and B, effects on the amplitude
of caffeine-induced Ca2+ responses. Bars show
means ± S.E. in noninjected control cells (open
bars) and in cells injected with 10 µM asRYR1
or asRYR2 or with a mixture of as1RYR1 + 2 or as1RYR1 + 2 + 3 at 10 µM each (A) or with 10 µM asRYR3
or scrambled (Scr) asRYR3 (filled
bars) (B). C, effects on the upstroke
velocity of caffeine-induced Ca2+ responses.
Bars show means ± S.E. in noninjected control cells
(open bars) or in cells injected with 10 µM as1RYR1, as1RYR2, as1RYR3, or a mixture of as1RYR1 + 2 (filled bars). The number of cells tested is
indicated in parentheses. Cells were obtained from six
different batches. , values significantly different from those
obtained in noninjected cells.
, value in as1RYR1 + 2-injected
cells significantly different from that in as1RYR3-injected cells.
Myocytes were loaded with fluo 3-AM.
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Fig. 5.
Effects of RYR3 antisense oligonucleotides on
Ca2+ responses evoked by flash photolysis of caged
Ca2+ in 10 mM
[Ca2+]o. Shown are typical Ca2+
transients evoked by UV flashes of 66 J in control cells in 1.7 mM [Ca2+]o (A) or 10 mM [Ca2+]o (B) and in a
cell injected with 10 µM as2RYR3 and superfused in
10 mM [Ca2+]o (C). Traces
show (from top to bottom) line scan fluorescence
image and averaged fluorescence from a 2-µm region (indicated by the
small vertical bar) of the line scan
image. Myocytes were loaded with fluo 3-AM and caged Ca2+
(DMNP-EDTA, AM).
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Fig. 6.
Compiled data showing the effect of RYR
antisense oligonucleotides on the amplitude of Ca2+
responses evoked by flash photolysis (66 J) of caged
Ca2+. A, bars show means ± S.E. in
1.7 mM [Ca2+]o and in 10 [Ca2+]o in noninjected control cells in the
absence (open bars) or in the presence of 100 µM ryanodine (filled bar).
B, bars show means ± S.E. in 10 mM [Ca2+]o in noninjected cells
(open bars) and in cells injected with 10 µM as2RYR3, as2RYR1, as2RYR2, and a mixture of as2RYR1 + 2 (filled bars). Cells were obtained from four
different cell batches. , value in 10 mM
[Ca2+]o significantly different from that in 1.7 mM [Ca2+]o.
, values
significantly different from those obtained in noninjected cells in 10 mM [Ca2+]o. Cells were obtained from
five different batches. Myocytes were loaded with fluo 3-AM and caged
Ca2+ (DMNP-EDTA, AM).
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Fig. 7.
Ca2+ sensitivity of RYR subtypes
in 10 mM [Ca2+]o. A,
peak Ca2+ transients induced by flash-photolytic
Ca2+ jumps were measured from the entire line scan image
and plotted against U.V. flashes of increasing energy, in noninjected
control cells in the absence ( ) or in the presence of 100 µM ryanodine for 20 min (
), and in cells injected with
as2RYR3 (
) or as2RYR1 + 2 (
).
, value obtained in 1.7 mM [Ca2+]o in noninjected cells. Data
are means ± S.E. for 10-12 cells obtained from five different
batches. B, normalized curves obtained by plotting
(F/Fo)/max
(F/Fo) against flash intensity in
noninjected control cells (
) and in cells injected with as2RYR3
(
) or as2RYR1 + 2 (
). Fitted curves were
obtained with the Boltzmann equation. Myocytes were loaded with fluo
3-AM and DMNP-EDTA, AM.
1-adrenergic agonist) were increased
(Fig. 8A). The mean upstroke
velocity and amplitude of the phenylephrine-induced Ca2+
responses increased from 13.6 ± 2.4 (
F/Fo·s
1,
n = 14) to 27.9 ± 4.1 (n = 10)
and from 1.90 ± 0.14 (
F/Fo, n = 14) to 2.34 ± 0.08 (n = 20),
respectively, in 1.7 mM [Ca2+]o and
in 10 mM [Ca2+]o (Fig.
8B). In 1.7 mM [Ca2+]o,
inhibition of the RYR3 subtype by treatment with antisense
oligonucleotides had no significant effect on the amplitude and
upstroke velocity of phenylephrine-induced Ca2+ waves (Fig.
8B). In contrast, in 10 mM
[Ca2+]o, inhibition of RYR3 reduced both the
amplitude and the upstroke velocity of the phenylephrine-induced
Ca2+ waves (Fig. 8B). Interestingly, the
amplitude and upstroke velocity of the phenylephrine-induced
Ca2+ responses in RYR3-deficient cells pretreated in 10 mM [Ca2+]o were similar to those
obtained in control cells superfused in 1.7 mM
[Ca2+]o (Fig. 8B). Taken together,
these results suggest that the RYR3 is responsible for the enhancement
of the phenylephrine-induced Ca2+ wave in
Ca2+-overloaded cells.
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Fig. 8.
Effects of RYR3 antisense oligonucleotides on
Ca2+ responses induced by phenylephrine in 10 mM [Ca2+]o. A,
typical Ca2+ waves induced by 10 µM
phenylephrine in 1.7 mM [Ca2+]o
(a) or 1 h after increasing
[Ca2+]o to 10 mM (b).
Traces show (from top to bottom) phenylephrine
application and averaged fluorescence from a 2-µm region of the line
scan image. B, compiled data showing the effects of as1RYR3
oligonucleotide on the amplitude and upstroke velocity of
phenylephrine-induced Ca2+ responses. Phenylephrine was
applied at 10 µM. Bars show means ± S.E.
in 1.7 mM [Ca2+]o and in 10 mM [Ca2+]o, either in noninjected
cells (open bars) or in cells injected with 10 µM as1RYR3 (filled bars). The
number of cells tested is indicated in parentheses. Cells
were obtained from four different batches. , values significantly
different from those obtained in noninjected cells in 10 mM
[Ca2+]o. Myocytes were loaded with fluo
3-AM.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
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We thank N. Biendon for secretarial assistance.
![]() |
FOOTNOTES |
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* This work was supported by grants from Centre National de la Recherche Scientifique, Centre National des Etudes Spatiales, Région Aquitaine, Pôle Médicament-Santé and Association Française contre les Myopathies, France.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. Tel.: 33 5 57 57 12 31; Fax: 33 5 57 57 12 27; E-mail:
jean.mironneau@umr5017.u-bordeaux2.fr.
Published, JBC Papers in Press, January 9, 2001, DOI 10.1074/jbc.M005994200
2 J. L. Morel, C. Le Sénéchal, and J. Mironneau, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: RYR, ryanodine receptor; SR, sarcoplasmic reticulum; DMNP-EDTA, AM, 1-(4,5-dimethoxy-2-nitrophenyl)-EDTA, tetra(acetoxymethylester); fluo 3-AM, fluo 3- acetoxymethylester.
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REFERENCES |
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