Skeletal Muscle Type Ryanodine Receptor Is Involved in Calcium
Signaling in Human B Lymphocytes*
Yoshitatsu
Sei
§,
Kathleen L.
Gallagher
, and
Anthony S.
Basile¶
From the
Department of Anesthesiology, Uniformed
Services University of the Health Sciences and the ¶ Laboratory of
Bioorganic Chemistry, NIDDK, National Institutes of Health,
Bethesda, Maryland 20814-4799
 |
ABSTRACT |
The regulation of intracellular free
Ca2+ concentration ([Ca2+]i) in
B cells remains poorly understood and is presently explained almost
solely by inositol 1,4,5-triphosphate (IP3)-mediated Ca2+ release, followed by activation of a store-operated
channel mechanism. In fact, there are reports indicating that
IP3 production does not always correlate with the magnitude
of Ca2+ release. We demonstrate here that human B cells
express a ryanodine receptor (RYR) that functions as a Ca2+
release channel during the B cell antigen receptor (BCR)-stimulated Ca2+ signaling process. Immunoblotting studies showed that
both human primary CD19+ B and DAKIKI cells express a
565-kDa immunoreactive protein that is indistinguishable in molecular
size and immunoreactivity from the RYR. Selective reverse
transcription-polymerase chain reaction, restriction fragment length
polymorphism, and sequencing of cloned cDNA indicated that the
major isoform of the RYR expressed in primary CD19+ B and
DAKIKI cells is identical to the skeletal muscle type (RYR1). Saturation analysis of [3H]ryanodine binding yielded
Bmax = 150 fmol/mg of protein and Kd = 110 nM in DAKIKI cells. In
fluo-3-loaded CD19+ B and DAKIKI cells,
4-chloro-m-cresol, a potent activator of Ca2+
release mediated by the ryanodine-sensitive Ca2+ release
channel, induced Ca2+ release in a
dose-dependent and ryanodine-sensitive fashion. Furthermore, BCR-mediated Ca2+ release in CD19+
B cells was significantly altered by 4-chloro-m-cresol and
ryanodine. These results indicate that RYR1 functions as a
Ca2+ release channel during BCR-stimulated Ca2+
signaling and suggest that complex Ca2+ signals that
control the cellular activities of B cells may be generated by
cooperation of the IP3 receptor and RYR1.
 |
INTRODUCTION |
In all cells, calcium ions play a critical role in the regulation
of diverse cell activities, including gene expression, folding and
processing of proteins, exocytosis and endocytosis, cell cycle progression, motility, proliferation, and differentiation (1). The
importance of Ca2+ signaling has also been demonstrated in
the B cells that are responsible for humoral immunity (2). In the
process of the humoral immune response, antigen binding to the B cell
antigen receptor (BCR)1
stimulates B cells to proliferate and secrete antigen-specific antibodies. An increase in the intracellular free Ca2+
concentration ([Ca2+]i) is a critical regulatory
event in BCR-mediated signal transduction (3, 4). Modulation of the
rise in [Ca2+]i during B cell activation has also
been suggested to be a key mechanism for controlling BCR-mediated
signal transduction by secondary signals produced by B cell accessory
molecules (e.g. CD19, CD21, CD22, CD40, etc.) (2, 5, 6).
Furthermore, it has recently been demonstrated that the temporal
characteristics (i.e. transient or sustained) and the
amplitude of the Ca2+ signal are important in activating
specific transcription factors (i.e. NF-
B, c-Jun
N-terminal kinase, NFAT, etc.), which determine the type of gene
expression in the B cell system (7). Similarly, sustained calcium
signals are often associated with enhanced proliferative responses and
secretion of antibodies (4). Therefore, the regulation of
Ca2+ signaling determines the ultimate responses of B
cells, which specifically include proliferation, apoptosis, and
secretion of specific antibodies.
Despite the prominent role of Ca2+ in B cell activation,
the molecular mechanisms responsible for Ca2+ movement in B
cells are not clearly understood. To date, the regulation of
[Ca2+]i in the B cell system is still explained
almost solely by IP3-mediated mechanisms. Antigen binding
to the BCR induces a biphasic increase in [Ca2+]i
(8, 9). The initial, rapid phase of the BCR-stimulated increase in
[Ca2+]i is the result of Ca2+ release
from intracellular stores, whereas the subsequent, sustained elevation
of [Ca2+]i results from Ca2+ entry
through plasma membrane channels, as indicated by its abolition in the
absence of extracellular Ca2+ (8, 9). As suggested in other
types of non-excitable cells (10-12), BCR-mediated Ca2+
entry is hypothesized to be a consequence of the emptying of the
intracellular IP3-sensitive Ca2+ store
("capacitative" Ca2+ entry mechanism) (13). Thus,
BCR-stimulation generates IP3, which activates
Ca2+ release from IP3-sensitive
Ca2+ stores, causing Ca2+ channels to open the
so-called store-operated channel. However, there are reports indicating
that IP3 production does not always correlate with the
magnitude of Ca2+ release (9, 14). These findings suggest
that an IP3-insensitive Ca2+ store is involved
in BCR-mediated Ca2+ signaling in B cells.
This study suggests that the ryanodine receptor (RYR) may contribute to
the IP3-insensitive component of BCR-stimulated
Ca2+ signaling. The RYR was originally found in the
sarcoplasmic reticulum of skeletal muscle (type 1 receptor, RYR1) and
cardiac muscle (type 2 receptor, RYR2) (15-17). Ca2+
release from the sarcoplasmic reticulum through these receptors plays a
central role in regulating the contraction of skeletal and cardiac
muscle fibers. A third type of RYR (type 3 receptor, RYR3) has been
detected in specific regions of the brain, such as corpus striatum,
thalamus, and hippocampus (18, 19), and in human Jurkat T cells (20).
The present studies demonstrate that the ryanodine receptor detectable
in B cells is identical to the skeletal muscle type (RYR1). Moreover,
the receptor was found to function as a Ca2+ release
channel during BCR-stimulated Ca2+ signaling. Therefore,
upon BCR stimulation, B cells utilize at least two types of
Ca2+ release channels, the IP3 receptor and
RYR1, to generate highly elaborate Ca2+ signaling.
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MATERIALS AND METHODS |
Human B Cells, Cell Lines, and Tissue--
Buffy coats were
obtained from healthy blood donors at the National Institutes of Health
Blood Bank (Bethesda, MD). Peripheral blood mononuclear cells were
isolated by Ficoll-Hypaque density gradient centrifugation.
CD19+ B cells were purified from the peripheral blood
mononuclear cells using an antibody-coupled magnetic bead isolation
system (Dynal, Oslo, Norway). Cells were first incubated with
monoclonal anti-CD19 antibody (Pharmingen, San Diego, CA) for 30 min at
4 °C, followed by a wash with Hanks' balanced salt solution and
incubation with goat anti-mouse IgG-coated M450 Dynabeads (Dynal) for
15 min at 4 °C. Cells attached to the beads were then isolated after
three washes with Hanks' balanced salt solution. Epstein-Barr
virus-transformed B cells (DAKIKI cells) (TIB206, American Type Culture
Collection, Rockville, MD) (21) were cultured in RPMI 1640 medium
supplemented with 10% fetal calf serum (Hyclone Laboratories, Logan,
UT), 1.5 g/l sodium bicarbonate, 4.5 g/l glucose, 10 mM
HEPES, 2 mM L-glutamine, 1 mM
sodium pyruvate, 100 units of penicillin, and 100 µg/ml streptomycin (Quality Biological, Inc., Gaithersburg, MD). Cell cultures were incubated at 37 °C in a humidified chamber with 5% CO2.
Human skeletal muscle from the vastus lateralis muscle, most of which was utilized for histopathology and a caffeine/halothane contracture test for diagnosing susceptibility to malignant hyperthermia, was used
to obtain control cDNA and protein for RYR1.
Western Blot Analysis for RYR1 Protein--
Tissues or purified
cells were disrupted in disposable Dounce homogenizers in buffer
containing 50 mM Tris-HCl (pH 7.4), 100 mM
NaCl, 1% Chaps, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 25 µg/ml p-nitrophenyl guanidinobenzoate were then incubated
for 20 min at 4 °C. Following centrifugation at 14,000 × g for 15 min, the supernatants were collected and analyzed
for total protein (BCA protein assay kit, Pierce). The protein samples
(10-75 µg/lane) were separated by SDS-polyacrylamide gel
electrophoresis on a 10% Tris/glycine gel. After separation, the
proteins were transferred to a polyvinylidene difluoride membrane
(Millipore Corp., Bedford, MA) and then probed with monoclonal anti-RYR
antibodies (Affinity Bioreagents Inc., Golden, CO). Alkaline
phosphatase-conjugated monoclonal anti-rabbit IgG antibody (Sigma) was
used to detect the primary rabbit antibodies. Chemiluminescence
detection was performed using the alkaline phosphatase substrate CSPD
(Tropix Inc.).
RT-PCR Restriction Fragment Length Polymorphism
Analysis--
Total RNA was extracted using the acid guanidinium
thiocyanate/phenol/chloroform method as described previously (22) and reverse transcription performed on the first strand of cDNA using a
First-Strand cDNA synthesis kit (Amersham Pharmacia Biotech). Synthesized cDNA was then amplified by RT-PCR using the primers reported by Hakamata et al. (20) (upstream primer,
5'-dTTCATGCTGCTGTTTTATAAGGT-3'; and downstream primer,
5'-dCAGATGAAGCATTTGGTCTCCAT-3'). These primers recognize all three
isoforms of RYR, producing an ~1200-bp product from the 3'-region of
RYR1, RYR2, and RYR3 (20). PCR amplifications were carried out with 100 ng of each primer in a total volume of 100 µl. The reaction solution
contained 50 mM KCl, 10 mM Tris-HCl (pH 8.3),
1.5 mM MgCl2, 0.5 mM each dNTP, and
1.5 units of Taq plus Pwo polymerase (Boehringer
Mannheim). The PCR amplification conditions were 95 °C for 2 min,
followed by 40 cycles at 95 °C for 1 min, 55 °C for 2 min, and
68 °C for 3 min, followed by a 7-min extension at 68 °C in a DNA
thermal cycler (Perkin-Elmer). The RT-PCR products were then digested with the selected restriction enzymes HgaI, BsmI,
and ApaI to identify the RYR isoform. Based on the known
sequences of human RYR isoforms, HgaI cuts the amplified
1200-bp RYR1 product into 740-, 349-, and 71-bp fragments, but it does
not digest human RYR2 or RYR3. BsmI cuts only RYR2 and
produces 810- and 320-bp fragments. ApaI cuts only RYR3 to
make 949- and 157-bp fragments. The PCR products were digested at
37 °C for 1 h with 1-5 units of the restriction endonucleases.
The restriction fragments were then resolved by electrophoresis on a
1% agarose gel and visualized on a UV transilluminator.
Selective RT-PCR Using an Isoform-specific Primer--
The
cDNAs obtained from DAKIKI cells, human skeletal muscle, lung
(CLONTECH, Palo Alto, CA), and brain
(CLONTECH) were amplified by PCR using primer sets
that selectively amplify specific isoforms of the RYR. Using the same
downstream primer (5'-dCAGATGAAGCATTTGGTCTCCAT-3') and upstream primers
JBR1 (5'-dGACATGGAAGGCTCAGCTGCT-3'), JBR2 (5'-dAAGGAGCTCCCCACGAGAAGT-3'), and JBR3 (5'-dGAGGAAGAAGCGATGGTGTT-3') amplifies an ~1200-bp product from the 3'-region of RYR1, RYR2, and RYR3, respectively. The PCR amplification conditions were 95 °C
for 2 min, followed by 40 cycles at 95 °C for 1 min, 55 °C for 2 min, and 68 °C for 3 min, followed by a 7-min extension at 68 °C.
The RT-PCR products were resolved by electrophoresis on a 1%
agarose gel and visualized on a UV transilluminator.
Cloning of Partial cDNAs from B Cells and
Sequencing--
Total RNA was isolated from DAKIKI cells,
reverse-transcribed, and amplified by PCR. A middle portion of RYR1,
which contains a sequence that exhibits marked differences among the
RYR isoforms, was amplified using an upstream primer
(5'-dTGGGCCCAAGAGGACTTCGT-3') and a downstream primer
(5'-dAGCACCATGGACGCCTTGTG-3'). A PCR product ~1200 bp in size was
purified from a 1% agarose gel with a JETSORB gel extraction kit
(GENOMED Inc., Research Triangle Park, NC) and cloned into
pGEMR-T vector using a TA cloning system (Promega, Madison,
WI). A purified DNA was then sequenced by a method of cycle sequencing using a forward primer (5'-dTGCCTCCGTCATTGACAACA-3') and an automated DNA sequencer (Applied Biosystems Model 373A).
Ryanodine Binding--
A radioligand binding assay was performed
with [3H]ryanodine to determine a profile of ryanodine
binding to the RYR in DAKIKI cells using the tissue preparation
technique modified from Chen et al. (23). Cells from six
75-cm2 culture flasks (~2-3 × 108
cells) were harvested by centrifugation at 500 × g.
The pellet was resuspended in 1.5 ml of buffer A (25 mM
Tris/HEPES (pH 7.5), 5 mM dithiothreitol, 1 µg/ml
aprotinin, and 0.5 mM 4-(2-aminoethyl)benzenesulfonyl fluoride); transferred to a Potter-Elvehjem homogenizer with a Teflon
pestle; and then disrupted with 25 strokes. An additional 1.5 ml of
buffer containing buffer A plus 0.5 M sucrose, 0.3 M KCl, and 40 µM CaCl2 was added
to the homogenate and processed with another 25 strokes. The homogenate
was centrifuged at 20,000 × g for 20 min at
0-4 °C. The supernatant was retained, and the pellet was
rehomogenized and recentrifuged. The pellet was retained and kept at
0-4 °C, and the supernatants were combined and centrifuged at
150,000 × g for 90 min. After centrifugation, the P1-2
and P3 pellets were resuspended in assay buffer (10 mM
PIPES citrate (pH 7.5), 0.5 M KCl, 10 mM ATP,
and 800 µM CaCl2). The binding assay was
performed in polystyrene multiwell plates (1-ml maximum volume; Beckman
Instruments). Assays were performed in duplicate, with each tube
containing 50 µl of tissue preparation (500 and 180 µg of
protein/tube for P1-2 and P3, respectively), 50 µl of [3H]ryanodine (final concentrations ranging from 50 to
600 nM; specific activity = 61.9 Ci/mmol; NEN Life
Science Products), 50 µl of unlabeled ryanodine (for determination of
nonspecific binding, final concentration = 10 µM),
and sufficient assay buffer to make a final volume of 200 µl. The
[3H]ryanodine was isotopically diluted to a specific
activity of 6.19 Ci/mmol using unlabeled ryanodine. The assay was
incubated in the dark at 37 °C for 90 min. It was then terminated by
vacuum filtration onto glass-fiber filters (Schleicher & Schuell No. 32) and washed with 2 ml of wash buffer (5 mM Tris-HCl, 0.5 M KCl, and 250 µM CaCl2) at
0-4 °C. Filters were placed into vials with 4 ml of CytoScint
scintillation fluid (ICN, Costa Mesa, CA) and counted for 5 min in a
scintillation counter (Beckman Instruments Model 6500).
Heavy sarcoplasmic reticulum fragments were prepared from frozen rabbit
skeletal muscle (Pel-Freez Biologicals, Rogers, AR) (24). Muscles were
ground and then homogenized in 15 ml of pyrophosphate buffer (20 mM sodium pyrophosphate, 20 mM
NaH2PO4, 1 mM MgCl2, and 0.5 mM EDTA (pH 7.4) at 0-4 °C) and 10% sucrose
using a Polytron (Brinkmann Instruments). The volume of the homogenate
was increased to 35 ml with cold buffer and centrifuged at 10,000 × g for 10 min at 0-4 °C. The supernatant was then
centrifuged at 10,000 × g for 15 min at 0-4 °C.
The pellet was resuspended in 15 ml of buffer and recentrifuged at
27,000 × g for 45 min at 0-4 °C. The final pellet
was resuspended in buffer and frozen at
70 °C overnight. Aliquots
(5 ml) were thawed, layered on a discontinuous sucrose gradient (4 ml
of 14% (w/v), 12 ml of 25%, 5 ml of 28%, 4 ml of 36%, and 3 ml of
45% sucrose in pyrophosphate buffer), and then centrifuged at
27,000 × g for 60 min at 4 °C. The material accumulating at the 36% sucrose layer was collected and resuspended in
assay buffer for the radioligand binding assay (~1 mg of protein/ml). [3H]Ryanodine binding to this preparation was performed
by adding 50 µl of tissue preparation to 50 µl of
[3H]ryanodine (final concentrations of 50-500
nM) and sufficient buffer to yield a 200-µl final volume.
The buffer was composed of 40 mM Tris-HCl (pH 7.4), 10 mM ATP, 800 µM CaCl2, 1.5 M KCl, and 200 mM sucrose (free calcium
concentration was set using "Bound and Determined" software,
obtained from K. B. Storey (25)). Nonspecific binding was determined
in the presence of 10 µM unlabeled ryanodine. The assay
was incubated for 60 min at 37 °C and terminated by rapid filtration
over a Brandel M-24R.
Measurement of Intracellular Calcium--
Changes in
[Ca2+]i were measured directly in human
peripheral blood mononuclear cells or DAKIKI cells by measuring the
fluorescence intensity of fluo-3-loaded cells (26, 27). Cells (2 × 106/ml) were loaded with 1 µM fluo-3/AM
(Molecular Probes, Inc., Eugene, OR) by incubation in subdued light (60 min, 25 °C) and stained with or without phycoerythrin-conjugated
monoclonal anti-CD19 antibody. Cells were then washed three times with
Hanks' balanced salt solution and resuspended in 1 ml of Hanks'
balanced salt solution and analyzed by FACScan (Becton-Dickinson, Palo
Alto, CA). The FL-1 signal for fluo-3 was calibrated by transporting in
saturating Ca2+ with either ionomycin or A23187 (Molecular
Probes, Inc.) to obtain the maximum signal
(Fmax) and then adding Mn2+ to
obtain the minimum signal (Fmin).
[Ca2+]i was calculated from the fluo-3
fluorescence intensity using the following formula:
[Ca2+]i = Kd(F
Fmin)/(Fmax
F), where [Ca2+]i = intracellular
ionized calcium concentration and Kd = 400 nM for the intracellular dye. For each experiment, the
fluo-3-loaded cells were analyzed to obtain an unstimulated base line.
The percentage of responding FL-1+ cells was then
calculated and analyzed.
 |
RESULTS |
The RYR Is Expressed at the Protein Level in Human Primary B Cells
and a B Cell Line (DAKIKI)--
SDS-polyacrylamide gel electrophoresis
immunoblot analysis with monoclonal anti-RYR antibody (clone 34-C)
revealed the presence of immunoreactive protein (565 kDa) in human
skeletal muscle tissues. An immunoreactive protein of the same size was
detected in both CD19+ B cells isolated from human
mononuclear cells and DAKIKI cells (Fig.
1A).

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Fig. 1.
A, Western blot analysis of the RYR.
First and second lanes,10 µg of total protein
from skeletal muscle samples from two individuals (Muscle-1
and Muscle-2); third lane,75 µg of protein from
primary human CD19+ B cells; fourth lane, 75 µg of protein from DAKIKI B cells. The protein preparation from the
second skeletal muscle sample (second lane,
Muscle-2) indicates partial degradation of the protein. This
blot is representative of a total of five experiments. Molecular size
markers are not included. B, agarose gel electrophoresis of
RYR RT-PCR products. Following RT-PCR, the 1200-bp products were
digested with HgaI, BsmI, or ApaI to
identify the isoforms of the RYR. Molecular size markers are shown in
the first lane. C, agarose gel electrophoresis of
amplicons following selective RT-PCR. cDNA obtained from DAKIKI
cells, skeletal muscle (S. Muscle), lung, and brain was
amplified using primer sets that specifically amplify RYR1, RYR2, or
RYR3.
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The RYR mRNA Detectable in Human Primary B Cells and a B Cell
Line (DAKIKI) Is Similar to Skeletal Muscle-type RYR1--
A PCR-based
restriction fragment length polymorphism method was designed to
identify isoforms of human RYR (RYR1, RYR2, and RYR3). In this method,
cDNA for RYR is synthesized from RNA by reverse transcription and
amplified by RT-PCR using the primers reported by Hakamata et
al. (20). The primers recognize all three isoforms of RYR,
producing an ~1200-bp product from the 3'-region of RYR1, RYR2, and
RYR3. To identify which isoforms were produced from B cell cDNA,
the RT-PCR products were digested with the selected restriction enzymes
HgaI, BsmI, and ApaI. Based on the
sequences of the human RYR available in the GenBankTM Data
Bank, HgaI, BsmI, and ApaI cut at a
unique site in the amplified sequences of RYR1, RYR2, and RYR3,
respectively. The majority of the PCR products from human skeletal
muscle were digested by HgaI, suggesting that the major
isoform of the RYR expressed in skeletal muscle is RYR1. Although the
overlapping of any ApaI-digested fragment by excess PCR
product prevents detection of possible ApaI digestion of
RYR3, incomplete HgaI digestion may be due to the presence
of RYR3, as suggested by a selective RT-PCR experiment (Fig.
1C). As clearly shown in Fig. 1B, primary B cells
and DAKIKI cells were completely digested by HgaI, but not
by ApaI or BsmI. This result suggests that the
major isotype of the RYR expressed in primary B cells and DAKIKI cells
is RYR1.
Expression of the RYR isoform was further investigated by selective
RT-PCR using isoform-specific primers (Fig. 1C). As
previously reported (28), RYR1, RYR2, and RYR3 mRNAs were highly
expressed in skeletal muscle, lung, and brain, respectively. RYR1 was
also expressed in lung, brain, and DAKIKI B cells. RYR2 was also
expressed in brain, as previously shown (28). Although at lower levels than RYR1, RYR3 was also detected in skeletal muscle. In human DAKIKI B
cells, neither the type 2 nor the type 3 isoform was expressed. The
results indicate that RYR1 is almost exclusively expressed in DAKIKI B
cells, thus confirming the restriction fragment length polymorphism result.
A middle portion of the RYR cDNA from DAKIKI cells was cloned into
pGEM-T vector and sequenced. The cloned cDNA contains an open
reading frame of 120 amino acid residues that corresponds to the
sequence of RYR1 (amino acids 2298-2417) and has a sequence that
exhibits marked differences among the RYR isoforms (human RYR1, amino
acids 2379-2417; human RYR2, amino acids 2347-2385; and rabbit RYR3,
amino acids 2248-2286) (Fig. 2). The
amino acid sequence from DAKIKI cells is identical to that of human
RYR1, except for one amino acid (Lys2323/Asn) (Fig. 2).
However, Gillard et al. (29) have reported that Lys2323 is an error in the sequence originally reported
(17) and corrected it to asparagine. Overall results suggest that RYR1
mRNA is expressed in human B cells.

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Fig. 2.
A, nucleotide and deduced amino acid
sequences of the RYR expressed in DAKIKI cells. The amino acid numbers
indicated correspond to human RYR1. B, alignment of the B
cell (B) RYR (from DAKIKI cells) with human (H)
RYR1, human RYR2, and rabbit (R) RYR3. Note that
Lys2323 in RYR1 is an error in the sequence originally
reported (17) and has been corrected to Asn (29). The cDNA sequence
of DAKIKI B cells, including those that are highly polymorphic among
the known three isoforms, is identical to that of human RYR1.
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[3H]Ryanodine
Binding--
[3H]Ryanodine bound to the P1-2 fraction of
the DAKIKI cell line with Kd = 130 ± 24 nM and Bmax = 170 ± 31 fmol/mg of protein (n = 3) (Fig.
3). The signal/noise ratio at 50 nM [3H]ryanodine was 1.2:1. There was no
specific binding of [3H]ryanodine to the P3 fraction of
the cell line at protein concentrations up to 180 µg/tube. In
contrast, [3H]ryanodine bound to receptors in rabbit
skeletal muscle preparations (in the presence of ATP) with
Kd = 40 ± 6.9 nM and
Bmax = 3.3 ± 0.5 pmol/mg of protein
(n = 5). The signal/noise ratio at 50 nM
[3H]ryanodine was 2.4:1.

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Fig. 3.
Representative binding isotherm of
[3H]ryanodine binding to a P1-2 fraction prepared from
DAKIKI cells. The equilibrium binding constants obtained using
nonlinear regression analysis (Prism, GraphPAD Software For Science,
San Diego, CA) for this presentation are Kd = 110 nM and Bmax = 150 fmol/mg of
protein. Inset, the Scatchard-Rosenthal plot of the data is
consistent with a single binding site. The Kd and
Bmax values obtained using linear regression
analysis are 160 nM and 190 fmol/mg of protein,
respectively. B/F, bound/free.
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4-Chloro-m-cresol (4-CmC) induced Ca2+ Release in
CD19+ B Cells in a Dose-dependent and
Ryanodine-sensitive Manner--
The RYR stimulator (30-32) 4-CmC (100 µM to 1 mM) caused a
dose-dependent increase in [Ca2+]i
(from ~50 nM to a maximum of 500 nM by 1 mM 4-CmC) in CD19+ B cells. The rise in
[Ca2+]i lasted for ~300-400 s after the
addition of 4-CmC (Fig. 4, A
and B). This increase was not totally blocked by the addition of excess EGTA (5 mM), indicating that 4-CmC
induced Ca2+ release and influx (Fig. 4, A and
B). Cells treated with ryanodine (200 µM)
exhibited significantly higher and longer rises in
[Ca2+]i induced by 4-CmC (Fig. 4, A
and B). 4-CmC also increased [Ca2+]i
in DAKIKI B cells in a dose-dependent and
ryanodine-sensitive manner (data not shown).

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Fig. 4.
Changes in [Ca2+]i
following treatment with 4-CmC (200 (A) and
500 (B) µM) in
CD19+ B cells. Fluo-3-loaded cells were stained with
phycoerythrin-conjugated monoclonal anti-CD19 antibody as described
under "Materials and Methods." Fluo-3 fluorescence in
CD19+ B cells was analyzed every 30 s after the
addition of 4-CmC with a FACScan. Cells were untreated ( ) or treated
with ryanodine (200 µM) for 30 min in
Ca2+-containing medium ( ) or with 5 mM EGTA
for 5 min prior to the addition of 4-CmC ( ).
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F(ab)2 Anti-IgM-induced Ca2+ Release Is
Altered by Ryanodine or 4-CmC in CD19+ B Cells--
After
the addition of EGTA (5 mM), F(ab)2 anti-IgM
induced a rapid and short-lasting increase in
[Ca2+]i in CD19+ B cells.
[Ca2+]i returned to base-line levels within
120 s (Fig. 5A). Cells
treated with ryanodine (200 µM) exhibited significantly larger Ca2+ release. The [Ca2+]i at
60 s after anti-IgM stimulation was significantly higher in cells
treated with ryanodine than in control cells (475 ± 43 nM (n = 5) and 270 ± 32 nM (n = 5), respectively; p < 0.01) (Fig. 5C). Following F(ab)2 anti-IgM
stimulation, subsequent stimulation with 4-CmC (1 mM)
induced a small Ca2+ release, and this release was smaller
in cells treated with ryanodine (Fig. 5A). In
Ca2+-free medium, the F(ab)2 anti-IgM-induced
increase in [Ca2+]i in CD19+ B cells
was significantly reduced by pre-exposing cells to 4-CmC (100 µM) (Fig. 4B). The
[Ca2+]i at 60 s after anti-IgM stimulation
was significantly lower in cells previously exposed to 4-CmC treatment
than in control cells (192 ± 28 nM (n = 3) and 305 ± 35 nM (n = 4),
respectively; p < 0.05) (Fig. 5C).

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Fig. 5.
Effects of ryanodine and 4-CmC treatment on
F(ab)2 anti-IgM-stimulated increases in
[Ca2+]i in CD19+ B cells.
A, changes in [Ca2+]i after
cross-linking membrane IgM with F(ab)2 anti-IgM followed by
4-CmC (1 mM) treatment in control cells ( ) and in cells
treated with ryanodine ( ). Cells were incubated with and without
ryanodine (200 µM) for 30 min before the addition of
anti-IgM. EGTA (5 mM) was added to the medium 5 min before
the cross-linking. B, changes in
[Ca2+]i following F(ab)2 anti-IgM
stimulation in control cells ( , , , ) and in cells treated
with 4-CmC (×, , +). Cells were incubated in
Ca2+-free (5 mM EGTA-containing) medium with or
without 4-CmC (100 µM). C,
[Ca2+]i at 60 s after F(ab)2
anti-IgM stimulation in CD19+ B cells in
Ca2+-free medium. Prior to the anti-IgM stimulation, cells
were treated either with ryanodine (200 µM) for 30 min in
Ca2+-containing medium (Exp. 1) or with 4-CmC
(100 µM) for 10 min in Ca2+-free medium
(Exp. 2). Data are represented as the mean ± S.E.
(n = 3-5; *, p < 0.05; **,
p < 0.01).
|
|
 |
DISCUSSION |
Although an IP3-insensitive Ca2+ store has
been suggested to be involved in BCR-mediated Ca2+
signaling in B cells (9, 14), neither the status of expression nor the
function of the RYR has been studied in human B cells. In this report,
we show that human B cells express a functional RYR that is likely
involved in BCR-mediated Ca2+ signaling in B cells.
Interestingly, unless an alternative splicing variant exists, selective
RT-PCR, restriction fragment length polymorphism, cloning, and cDNA
sequence analysis indicate that the major isoform of the RYR expressed
in B cells is a type 1 isoform of RYR that was originally recognized to
be abundantly expressed in skeletal muscle (33).
Considering our current understanding of the functional properties of
the isoforms, it is interesting that non-excitable B cells express RYR1
rather than RYR3. RYR1 in skeletal muscle is activated by direct
physical coupling to the voltage-sensitive dihydropyridine receptor,
whereas RYR3 is activated by cyclic ADP-ribose, which may act as a
second messenger for intracellular Ca2+ signaling in
non-muscle cells (1, 34). However, because the L-type
dihydropyridine-sensitive Ca2+ channel has recently been
suggested to be involved in anti-Ig-induced Ca2+ influx in
rat B lymphocytes (35), it may be important to investigate whether the
dihydropyridine-sensitive Ca2+ channel is expressed and
associated with RYR1 in human B cells.
Pharmacological studies indicate that the RYR expressed in B cells
functions as a Ca2+ channel. 4-CmC, a RYR stimulator
observed in skeletal muscle (30-32), increased
[Ca2+]i in a dose-dependent fashion
in both primary CD19+ (or CD21+) B cells and
DAKIKI cells. That 4-CmC-induced increases in
[Ca2+]i result largely from Ca2+
release from intracellular Ca2+ stores was indicated by the
maintenance of [Ca2+]i in the absence of
extracellular Ca2+. Moreover, in the presence of 200 µM ryanodine, 4-CmC induced a higher maximum and slower
decay in [Ca2+]i than in the control. These
results may be related to the findings in skeletal muscle that
ryanodine keeps the Ca2+ release channel of the
intracellular Ca2+ stores in an open state (36), thus
maintaining RYR-mediated elevations in [Ca2+]i.
However, in skeletal muscle, high affinity ryanodine-binding sites
(Kd
1-5 nM) lock the
Ca2+ channel in an open state, whereas low affinity sites
(Kd
30-80 nM) inhibit channel
opening (34, 37). Accordingly, nanomolar to low micromolar
concentrations of ryanodine (<10 µM) tend to activate
Ca2+ release, whereas higher concentrations (>200
µM) block the Ca2+ channel in skeletal muscle
(34, 37).
B cells were found to have a single affinity site for
[3H]ryanodine with a Kd of 110 nM. Although this appears to be similar to the low affinity
site in skeletal muscle, it behaves like the high affinity site. This
may be due to differences in stoichiometry, localization of the
receptor, or association with endogenous effectors. Indeed,
pharmacological properties of RYR1, including
[3H]ryanodine binding, vary greatly in different cell
types (34, 37). RYR1 in skeletal muscle is well known to be abundant at the triad junction; physically attached to the voltage-sensitive Ca2+ channel localized in the transverse tubular membrane;
and associated with triadin, calmodulin, and FK506-binding protein (34,
37). However, neither localization of the receptor nor its association with other modulatory proteins is known in B lymphocytes.
Findings by other investigators have already suggested that an
IP3-insensitive Ca2+ store is involved in
BCR-mediated Ca2+ signaling in human B lymphocytes. For
example, following BCR stimulation, increases in
[Ca2+]i of 25-35% of control levels were not
affected when IP3 production was abolished by tyrosine
kinase inhibition (14). Similarly, inositol monophosphate generation
did not correlate well with changes in Ca2+ in human B
cells when these two parameters were compared over different types of
stimulation, i.e. cross-linking membrane IgG, IgD, and IgM
(9). Cross-linking membrane IgD induced the largest release of
Ca2+, but the inositol monophosphate production was the lowest.
In addition to the molecular biological evidence of expression of RYR1
in human B cells, pharmacological studies suggest that the release of
Ca2+ from stores through the RYR is involved in
BCR-mediated Ca2+ signaling. Similar to its effects on
4-CmC-induced Ca2+ release, F(ab)2 anti-IgM in
the presence of ryanodine induced a higher maximum and a slower decay
in [Ca2+]i in Ca2+-containing medium
(data not shown) and a higher maximum in Ca2+-free medium
than in the control (Fig. 5, A and C). These
results suggest that ryanodine maintains a Ca2+ release
channel in an open state, as demonstrated in skeletal muscle.
Furthermore, depleting Ca2+ in the 4-CmC-sensitive store
significantly decreased the magnitude of BCR-mediated Ca2+
release (Fig. 5, B and C), suggesting that B
cells utilize a RYR-operated Ca2+ store during BCR-mediated
activation perhaps in conjunction with the IP3 receptor.
B cells appear to decode diverse patterns of Ca2+ waves as
signals to determine their ultimate responses, such as proliferation, apoptosis, and secretion of specific antibodies (7). Such highly intricate patterns of Ca2+ waves may be generated by
cooperation of the IP3 receptor and RYR1 during
BCR-stimulated Ca2+ signaling. It is also possible that
RYR1 may play a specific role in B cell activation. In lymphocytes,
IP3 and the RYR coexist in murine T lymphoma BW5147 cells
(38) and human Jurkat T cells (20). In the BW5147 cells, a RYR that
exhibits only a single, low affinity
[3H]ryanodine-binding site (Kd = 200 nM) becomes associated with concanavalin A receptor-patched
and -capped structures following concanavalin A stimulation (38).
Similarly, Ono et al. (39) have reported that the
Epstein-Barr virus caused increases in [Ca2+]i in
a ryanodine-sensitive manner and was temporally associated with
Epstein-Barr virus receptor (CD21) capping in human B lymphocytes.
Despite the relatively low density of RYR expression, the much larger
Ca2+ conductance generated through the RYR (compared with
the IP3 receptor channel) may make it more effective in
producing the significant changes in Ca2+ required for
spatiotemporal phenomena such as movement of cellular components
(e.g. patching and capping) and secretion of Ig, hypotheses that are now under investigation.
 |
ACKNOWLEDGEMENTS |
We gratefully thank Drs. J. W. Daly,
S. M. Muldoon, M. Srivastava, and H. A. Pollard for helpful
discussions and critical comments. We also thank Dr. Hakamata for
providing a rabbit RYR3 sequence.
 |
FOOTNOTES |
*
This work was supported by Research Grant R08078 from the
Uniformed Services University of the Health Sciences (to Y. S.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF075460.
§
To whom correspondence should be addressed: Dept. of
Anesthesiology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Rd., Bethesda, MD 20814-4799. Tel.: 301-295-3167; Fax: 301-295-2200; E-mail: ysei{at}mx3.usuhs.mil.
 |
ABBREVIATIONS |
The abbreviations used are:
BCR, B cell antigen
receptor;
[Ca2+]i, intracellular free
Ca2+ concentration;
IP3, inositol
1,4,5-trisphosphate;
RYR, ryanodine receptor;
Chaps, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
RT-PCR, reverse transcription-polymerase chain reaction;
bp, base pair(s);
PIPES, 1,4-piperazinediethanesulfonic acid;
4-CmC, 4-chloro-m-cresol.
 |
REFERENCES |
-
Berridge, M. J.
(1997)
J. Physiol. (Lond.)
499,
291-306[Medline]
[Order article via Infotrieve]
-
Cambier, J. C.,
Pleiman, C. M.,
and Clark, M. R.
(1994)
Annu. Rev. Immunol.
12,
457-486[CrossRef][Medline]
[Order article via Infotrieve]
-
Baixeras, E.,
Guido, K.,
Cuende, E.,
Marquez, C.,
Boscá, L.,
Mártinez, J.,
and Mártinez, A.-C.
(1993)
Immunol. Rev.
132,
5-47[Medline]
[Order article via Infotrieve]
-
Yamada, H.,
June, C. H.,
Finkelman, F.,
Brunswick, M.,
Ring, M. S.,
Lees, A.,
and Mond, J. J.
(1993)
J. Exp. Med.
177,
1613-1621[Abstract]
-
Peaker, C. J. G.
(1994)
Curr. Opin. Immunol.
6,
359-363[CrossRef][Medline]
[Order article via Infotrieve]
-
Tedder, T. F.,
Zhou, L.-J.,
and Engel, P.
(1994)
Immunol. Today
15,
437-442[CrossRef][Medline]
[Order article via Infotrieve]
-
Delmetsch, R. E.,
Lewis, R. S.,
Goodnow, C. C.,
and Healy, J. I.
(1997)
Nature
386,
855-858[CrossRef][Medline]
[Order article via Infotrieve]
-
Bijsterbosch, M. K.,
Rigley, K. P.,
and Klaus, G. G. B.
(1986)
Biochem. Biophys. Res. Commun.
137,
500-506[Medline]
[Order article via Infotrieve]
-
Roifman, C. M.,
Mills, G. B.,
Stewart, D.,
Cheung, R. K.,
Grinstein, S.,
and Gelfand, E. W.
(1987)
Eur. J. Immunol.
17,
1737-1742[Medline]
[Order article via Infotrieve]
-
Hoth, M.,
and Penner, R.
(1992)
Nature
355,
353-356[CrossRef][Medline]
[Order article via Infotrieve]
-
Takemura, H.,
Hughes, A. R.,
Thastrup, O.,
and Putney, J. W., Jr.
(1989)
J. Biol. Chem.
264,
12266-12271[Abstract/Free Full Text]
-
Zweifach, A.,
and Lewis, R.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
1993,
6295-6299
-
Putney, J. W.
(1990)
Cell Calcium
11,
611-624[Medline]
[Order article via Infotrieve]
-
Padeh, S.,
Levitzki, A.,
Gazit, A.,
Mills, G. B.,
and Roifman, C. M.
(1991)
J. Clin. Invest.
87,
1114-1118[Medline]
[Order article via Infotrieve]
-
Otsu, K.,
Willard, H. F.,
Khanna, V. K.,
Zorzato, F.,
Green, N. M.,
and MacLennan, D. H.
(1990)
J. Biol. Chem.
265,
13472-13483[Abstract/Free Full Text]
-
Takeshima, H.,
Nishimura, S.,
Matsumoto, T.,
Ishida, H.,
Kangawa, K.,
Minamino, N.,
Matsuo, H.,
Ueda, M.,
Hanaoka, M.,
Hirose, T.,
and Numa, S.
(1989)
Nature
339,
439-445[CrossRef][Medline]
[Order article via Infotrieve]
-
Zorzato, F.,
Fujii, J.,
Otsu, K.,
Phillips, M.,
Green, N. M.,
Lai, F. A.,
Meissner, G.,
and MacLennan, D. H.
(1990)
J. Biol. Chem.
265,
2244-2256[Abstract/Free Full Text]
-
Hakamata, Y.,
Nakai, J.,
Takeshima, H.,
and Imoto, K.
(1992)
FEBS Lett.
312,
229-235[CrossRef][Medline]
[Order article via Infotrieve]
-
Takeshima, H.,
Nishimura, S.,
Nishi, M.,
Ikeda, M.,
and Sugimoto, T.
(1993)
FEBS Lett.
322,
105-110[CrossRef][Medline]
[Order article via Infotrieve]
-
Hakamata, Y.,
Nishimura, S.,
Nakai, J.,
Nakashima, Y.,
Kita, T.,
and Imoto, K.
(1994)
FEBS Lett.
352,
206-210[CrossRef][Medline]
[Order article via Infotrieve]
-
Millet, I.,
Samarut, C.,
and Revillard, J.-P.
(1988)
Eur. J. Immunol.
18,
545-550[Medline]
[Order article via Infotrieve]
-
MacDonald, R. J.,
Swift, G. H.,
Przybyla, A. E.,
and Chirgwin, J. M.
(1987)
Methods Enzymol.
152,
219-227[Medline]
[Order article via Infotrieve]
-
Chen, W.,
Vaughan, D. M.,
Airey, J. A.,
Coronado, R.,
and MacLennan, D. H.
(1993)
Biochemistry
32,
3743-3753[Medline]
[Order article via Infotrieve]
-
Campbell, K. P.,
and MacLennan, D. H.
(1981)
J. Biol. Chem.
256,
4626-4632[Abstract/Free Full Text]
-
Brooks, S. P. J.,
and Storey, K. B.
(1992)
Anal. Biochem.
201,
119-126[Medline]
[Order article via Infotrieve]
-
Sei, Y.,
and Arora, P.
(1991)
J. Immunol. Methods
137,
237-244[CrossRef][Medline]
[Order article via Infotrieve]
-
Sei, Y.,
Takemura, M.,
Gusovsky, F.,
Skolnick, P.,
and Basile, A.
(1995)
Exp. Cell Res.
216,
222-231[CrossRef][Medline]
[Order article via Infotrieve]
-
Gianni, G.,
Conti, A.,
Mammarella, S.,
Scrobogna, M.,
and Sorrentino, V.
(1995)
J. Cell Biol.
128,
893-904[Abstract]
-
Gillard, E. F.,
Otsu, K.,
Fuji, J.,
Duff, C.,
Leon, S. D.,
Khanna, V. K.,
Britt, B. A.,
Worton, R. G.,
and MacLennan, D. H.
(1992)
Genomics
13,
1247-1254[Medline]
[Order article via Infotrieve]
-
Herrmann-Frank, A.,
Richter, M.,
Sarkozi, S.,
Mohr, U.,
and Lehmann-Horn, F.
(1996)
Biochim. Biophys. Acta.
1289,
31-40[Medline]
[Order article via Infotrieve]
-
Herrmann-Frank, A.,
Richter, M.,
and Lehmann-Horn, F.
(1996)
Biochem. Pharmacol.
52,
149-155[CrossRef][Medline]
[Order article via Infotrieve]
-
Zorzato, F.,
Scutari, E.,
Tegazzin, V.,
Clementi, E.,
and Treves, S.
(1993)
Mol. Pharmacol.
44,
1192-1201[Abstract]
-
Inui, M.,
Saito, A.,
and Fleischer, S.
(1987)
J. Biol. Chem.
262,
1740-1747[Abstract/Free Full Text]
-
Zucchi, R.,
and Ronca-Testoni, S.
(1997)
Pharmacol. Rev.
49,
1-51[Abstract/Free Full Text]
-
Akha, A. A. S.,
Willmott, N. J.,
Brickley, K.,
Dolphin, A. C.,
Galione, A.,
and Hunt, S. V.
(1996)
J. Biol. Chem.
271,
7297-7300[Abstract/Free Full Text]
-
Imagawa, T.,
Smith, J. S.,
Coronado, R.,
and Campbell, K. P.
(1987)
J. Biol. Chem.
262,
16636-16643[Abstract/Free Full Text]
-
Coronado, R.,
Morrissette, J.,
Sukhareva, M.,
and Vaughan, D. M.
(1994)
Am. J. Physiol.
266,
C1485-C1504[Abstract/Free Full Text]
-
Bourguignon, L. Y. W.,
Chu, A.,
Jin, H.,
and Brandt, N. R.
(1995)
J. Biol. Chem.
270,
17917-17922[Abstract/Free Full Text]
-
Ono, A.,
Tatsumi, H.,
Yamamoto, K.,
and Katayama, Y.
(1995)
Bull. Tokyo Med. Dent. Univ.
42,
9-18[Medline]
[Order article via Infotrieve]
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