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*
Department of Anesthesiology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814;
Department of Medical Technology, School of Medical Sciences, University of Tokushima, Tokushima, Japan; and
Fujisaki Cell Center, Hayashibara Biochemical Laboratories, Inc., Okayama, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(MIP1) or TGF-
. Type 3 RYR
(RYR3) was not detected in PBMCs. Many hemopoietic cell lines expressed
not only RYR1 or RYR2 but also RYR3. The expression of the isoforms was
not associated with specific cell lineage. We showed that the
RYR-stimulating agent 4-chloro-m-cresol (4CmC) induced
Ca2+ release and thereby confirmed functional expression of
the RYR in the cell lines expressing RYR mRNA. Moreover, concordant
induction of RYR mRNA with Ca2+ channel function was found
in Jurkat T cells. In untreated Jurkat T cells, 4CmC (>1 mM) had no
effect on Ca2+ release, whereas 4CmC (<400 µM) caused
Ca2+ release after the induction of RYR2 and RYR3 that
occurred after treatment with stromal cell-derived factor 1,
macrophage-inflammatory protein-1, or TGF-. Our results
demonstrate expression of all three isoforms of RYR mRNA in hemopoietic
cells. Induction of RYRs in response to chemokines and TGF- suggests
roles in regulating Ca2+-mediated cellular responses during
the immune response. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B, JNK, NF-AT, etc.), and thus in a variety of
later events in the immune cell activation (4). Therefore,
the regulation of Ca2+ signaling determines the
ultimate response of an immune cell.
An early manifestation of mitogen- or cell surface receptor-stimulated
immune cell activation is a biphasic increase in
[Ca2+]i which is the
result of rapid Ca2+ release from intracellular
stores followed by sustained Ca2+ influx through
store-operated Ca2+ channels (SOC) (5, 6). Ca2+ release from intracellular stores
is consequent to inositol 1,4,5-trisphosphate
(IP3) formation. The activation of multiple
protein tyrosine kinases occurs immediately after surface receptor
ligation (7, 8). Phospholipase C
is then recruited to
an upstream tyrosine kinase via its SH2 domains and activated by
phosphorylation. Phospholipase C activation leads to the hydrolysis
of phosphatidylinositol 4,5-bisphosphate, yielding
IP3 and diacyl glycerol.
IP3 then mediates the activation of
Ca2+ release from stores in the endoplasmic
reticulum through the IP3 receptor. Therefore,
calcium mobilization after receptor cross-linking in the immune cells
has been explained almost solely by IP3-mediated
mechanisms.
Although IP3 is a key messenger regulating [Ca2+]i, recent studies have postulated the possibility that the ryanodine receptor (RYR) contributes to the IP3-insensitive component of Ca2+ signaling in immune cells (9, 10, 11, 12). The RYR was originally found in the sarcoplasmic reticulum of skeletal muscle (type 1 receptor; RYR1) and cardiac muscle (type 2 receptor; RYR2) (13, 14, 15). 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, nonmuscle tissues, and also skeletal muscle (16, 17, 18). We recently demonstrated that human B cells express a RYR that is identical with skeletal muscle type I by RFLP studies and sequencing analysis of partially cloned cDNA (12). In addition, 4-chloro-m-cresol (4CmC), a potent activator of the RYR (19), induced Ca2+ release after depleting IP3-sensitive Ca2+ pools in B cells (12). These results suggested that human B cells express functional RYR1 that is involved in regulating Ca2+ signaling, perhaps in conjunction with the IP3 receptor. For T cells, expression of the RYR has been found in human Jurkat T cells (10, 11) and murine T lymphoma cells (9). In both T cell lines, cyclic ADP-ribose increased [3H]ryanodine binding and induced Ca2+ release from intracellular Ca2+ stores (9, 10). The isoform of the RYR expressed in Jurkat T cells was identified to be type 3 (10, 11). Therefore, the RYR3 has been proposed to control [Ca2+]i in response to cyclic ADP-ribose during T cell activation (10). These findings of the RYR in T and B cells allowed us to hypothesize that the RYRs are more widely expressed and responsible for regulating [Ca2+]i in immune cells than currently thought.
In this study, we have investigated expression of all three isoforms of the RYR in human primary T cells, B cells, and monocytes using selective RT-PCR followed by RFLP analysis. We also examined a total of 164 human hemopoietic cell lines (36 T cell, 92 B cell, 19 myelomonocytic, 11 megakaryocytic, 3 erythrocytic, and 3 nonlymphocytic, nonmyelocytic) to determine the lineage and differentiation specificity of the expression of the 3 RYRs. The possibility that any isoform of RYR is induced by stimulation with mitogens, chemokines, and other stimuli were investigated to gain insight of the roles of this Ca2+ release channel in immune function. Finally, to verify the functional expression of the RYRs, Ca2+ release by RYR-stimulating agents was assessed using the cell lines expressing the RYR mRNA. A global view of RYR expression in human immune cells was addressed in this study.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Stroma-derived factors 1 and 1 (SDF-1 and -1),
macrophage-inflammatory protein-1 (MIP1), TGF-, RANTES, nerve
growth factor, and 4CmC were obtained from Calbiochem (San Diego, CA).
PHA, Con A (type IV), LPS (Escherichia coli; B5W), PMA, and
caffeine were from Sigma (St. Louis, MO). Fluo-3 acetoxymethyl ester
was obtained from Molecular Probes (Eugene, OR). Anti-CD19 and
anti-CD3 mAbs were from BD PharMingen (San Diego, CA). Total RNA
isolated from normal heart and mRNA from brain was purchased from
Invitrogen (Carlsbad, CA).
Primary cells, cell lines, and tissues
Buffy coats were obtained from healthy blood donors at the National Institutes of Health Blood Bank (Bethesda, MD). PBMCs were isolated by Ficoll-Hypaque density gradient centrifugation. CD3+ T cells, CD19+ B cells, and CD14+ monocytes were purified from the PBMCs using an Ab-magnetic bead isolation system (Dynal, Oslo, Norway). Cells (107 cells) were first incubated with Dynabeads coated with anti-CD3 mAb for 30 min at 4°C. CD3+ cells attaching to the beads were isolated after three washes with HBSS. Unattached cells were then incubated with anti-CD19 beads for 30 min at 4°C. CD19+ cells were isolated after three washes with HBSS, and unattached cells were incubated with anti-CD14 beads for 30 min at 4°C. CD14+ cells were then isolated after three washes with HBSS. Jurkat, SupT1, H9, CEM, SKW6.4, DAKIKI, THP-1, and U937 were obtained from American Type Culture Collection (ATCC; Manassas, VA). Other cell lines used in this study were from the repository at Fujisaki Cell Center (Okayama, Japan) (20). Cells were cultured in RPMI 1640 supplemented with 10% FCS (HyClone, Logan, UT), 2 mM L-glutamine, 100 U penicillin, and 100 µg/ml streptomycin (Quality Biological, Gaithersburg, MD). Cell cultures were incubated at 37°C in a humidified chamber with 5% CO2. Anonymous tissue sample from the vastus lateralis muscle, most of which was used for histopathology and caffeine/halothane contracture testing for diagnosing susceptibility to malignant hyperthermia, was used to obtain control cDNA and protein for the 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% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 25 µg/ml p-nitrophenylguanidinobenzoate and then incubated for 20 min at 4°C. After centrifugation at 14,000 x g for 15 min, the supernatants were collected and analyzed for total protein (BCA protein assay kit; Pierce, Rockford, IL). The protein samples (10–75 µg/lane) were separated using SDS-PAGE on a 10% Tris-glycine gel. After separation, the proteins were transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA), and then probed with monoclonal anti-RYR Abs (Affinity Bioreagents, Golden, CO). Alkaline phosphatase-conjugated monoclonal anti-rabbit IgG Ab (Sigma) was used to detect the primary rabbit Abs. Chemiluminescence detection was performed using the alkaline phosphatase substrate CSPD (Tropix, Bedford, MA).
Selective RT-PCR followed by RFLP analysis
Total RNA was extracted using the SV Total RNA Isolation System
(Promega, Madison, WI), and reverse transcription was performed to the
first strand of cDNA using a cDNA synthesis kit (Promega). Synthesized
cDNA was then amplified by RT-PCR using a primer set which selectively
amplifies specific isoform of the RYR. Using the same downstream
primer, 5'-dC-AGATGAAGCATTTGGTCTCCAT-3', and an isoform-specific
upstream primer: JBR1, 5'-dG-ACATGGAAGGCTCAGCTGCT-3'; JBR2,
5'-dAAGGAGCTCCCCACGAGAAGT-3'; and JBR3, 5'-dAAGAGGAAGAAGCGATGGT-3',
an 
1200-bp product was recognized from the 3'-regions of RYR1, RYR2,
and RYR3, respectively. PCR amplifications were conducted using the
Expand Long PCR system (Boehringer Mannheim, Indianapolis, IN). PCR was
performed in a 50-µl reaction mixture containing 100 ng DNA, 15 pmol
of each primer, 0.5 mM dNTPs, 2.5 U Expand Long polymerase mixture and
Expand Long PCR buffer 3 (Boehringer Mannheim). The PCR amplification
conditions were 95°C for 2 min, followed by 40 cycles of 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 then digested with
selected restriction enzymes, HgaI, BsmI, and
HindIII to identify the RYR isoform. Based on the known
sequences of human RYR isoforms, HgaI cuts the amplified
1112-bp RYR1 product into 692-, 349-, and 71-bp fragments, but it does
not digest human RYR2 or RYR3. BsmI cuts only the 1083-bp
RYR2 and produces 762- and 321-bp fragments. HindIII cuts
the 1015-bp RYR3 product to make 537- and 478-bp fragments. The PCR
products were digested at 37°C for 1 h with 1–5 U of the
restriction endonucleases. The restriction fragments were then resolved
by electrophoresis on a 2% agarose gel and visualized on a UV
transilluminator. As a control of mRNA input, -actin mRNA levels
were determined for each sample in separate RT-PCR. For -actin
amplification, PCR was performed with 25 cycles to ensure that the
amplification was completed within the linear range. The sequences of
primers for -actin were 5'-dAAGAGAGGCATCCTCACCCT-3' (sense) and
5'-dTGCTGATCCACATCTGCTGGA-3' (antisense). In some experiments, the
signal ratio of RYR to -actin was determined on the basis of the
ratio of the intensity of the PCR product compared with the
corresponding -actin band. The PCR products were imaged, and the
relative OD of each band was measured and analyzed using NIH Image
software.
Ca2+ mobilization test using B cells
Relative changes in [Ca2+]i were derived from changes in the fluorescence intensity of fluo-3-loaded cells (21). Cells (2 x 106/ml) were loaded with 1 µM fluo-3 acetoxymethyl ester in subdued light (30 min, 25°C). Cells were then washed once with HBSS, resuspended in 1 ml of HBSS, and analyzed by FACScan (Becton Dickinson). Forward and right angle scatter signals were displayed on a linear scale, with the forward scatter adjusted to gate cells from debris. The fluo-3 fluorescence (excitation at 488 nm with emission at 525 nm) was detected after separation with a 530 (FL-1) band pass filter. FL-1 fluorescence was recorded, amplified, and displayed on a logarithmic scale. For each experiment, the fluo-3-loaded cells were analyzed to obtain an unstimulated baseline. Cells were then exposed to caffeine, ryanodine, or 4CmC and analyzed continuously at rates of 400-1000 cells/s. The percentage of fluo-3+ cells (or mean fluorescence channel of fluo-3) relative to unstimulated baseline was analyzed to monitor the magnitude of elevation in [Ca2+]i in cells using CellQuest software (Becton Dickinson).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Expression of the three isoforms of RYR mRNA was investigated by
selective RT-PCR followed by RFLP analysis. In this method, cDNA for
RYR is synthesized from mRNA by reverse transcription and amplified by
selective PCR using the isoform-specific primers. The isoform-specific
PCR primers for amplification of the human RYR1, RYR2, and RYR3 were
designed to produce an 1200-bp product from the 3'-region of the
RYR1, RYR2, and RYR3. To further confirm specificity of the RT-PCR
products, the PCR products were digested with selected restriction
enzymes HgaI, BsmI, and HindIII. Based
on the sequences of human RYR1, RYR2, and RYR3 available in the
GenBank, HgaI, BsmI, and HindIII cut
at a unique site in the amplified sequences for RYR1, RYR2, and RYR3,
respectively. We tested our two-step method by examining cDNAs from
skeletal muscle, cardiac muscle, and brain which have been shown to
express type 1, type 2, and type 3, respectively (Refs. 15
and 17 and Fig. 1
). As
predicted based on previous observations, RYR1 mRNA was highly
expressed in skeletal muscle. RYR3 mRNA was also detected at lower
levels than RYR1 in skeletal muscle (Fig. 1A). In cardiac
muscle, RYR2 mRNA was dominantly expressed compared with types 1 and 3
(Fig. 1A). In brain, all three isoforms were highly
expressed (Fig. 1A). Specificity of the selective RT-PCR
products was then examined by RFLP analysis. The RT-PCR products
amplified for the RYR1, RYR2, and RYR3 from skeletal muscle, cardiac
muscle, and brain, respectively, were digested with restriction enzymes
HgaI, BsmI, and HindIII. The PCR
product amplified for the RYR1 from skeletal muscle was cut into 692-,
349-, and 71-bp fragments by HgaI, but it was not digested
with BsmI or HindIII (Fig. 1B). The
amplicon for RYR2 from cardiac muscle was cut into 762- and 321-bp
fragments by BsmI (Fig. 1B). The product for RYR3
from brain was cut into 537- and 478-bp fragments only by
HindIII (Fig. 1B).
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and Table I). Type 1 RYR was detected in freshly
isolated CD19+ B lymphocytes from five of nine
donors. The RYR1 mRNA was less frequently detected in
CD3+ T lymphocytes and
CD14+ monocytes. Type 2 RYR was mainly detected
in CD3+ T cells. For type 3 amplification, we
frequently observed smear without a distinguishable single band of PCR
product, whereas RYR3 mRNA from control brain tissue was successfully
amplified in every experiment (Fig. 2). The smearing was not due to
overexpression of type 3 mRNA, because the product was never detected
even in samples diluted up to 104 times. Because
-actin was highly expressed in all cDNA samples applied for the RYR3
amplification, we concluded that type 3 RYR mRNA was not present at
detectable levels in peripheral T cell, B cell, and monocyte
populations from the donors that we examined. We performed SDS-PAGE
immunoblot analysis to examine the protein expression in mononuclear
cells from the donor who showed high levels of RYR1 mRNA in B cells and
monocytes. The immunoblot with monoclonal anti-RYR Ab (clone 34-C)
revealed the presence of the immunoreactive protein that was similar in
size to the protein from skeletal muscle in purified
CD19+ B cells and CD14+
monocytes (Fig. 3). Some immunoreactivity
was seen in purified CD3+ T cell and total
mononuclear cell preparation (Fig. 3).
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A large array of human hemopoietic cell lines at various
stages of differentiation allows us to determine the lineage and
differentiation specificity of the expression of genes and proteins. To
investigate specificity of expression of RYR isoforms, we examined
total 164 human cell lines, consisting 36 T and 92 B cell leukemia
lines and 19 myelomonocytic, 11 megakaryocytic, 3 erythrocytic, and 3
nonlymphocytic, nonmyelocytic cell lines (Tables I–III
). In T cell
lines, 22% of cell lines expressed type 1, 2, or 3 of the RYR mRNA.
All T cell lines expressing detectable levels of RYR mRNA were from
either category of blast-III or blast-IV (Table II). T cell lines classified in blast-III
and -IV are differentiated and blocked at a more mature stage than
cells in blast-I and -II (22). Nine cells classified in
blast-I or blast-II did not express RYR mRNA. Similarly, 21% of B cell
lines (mainly mature B cell and plasma cell types) expressed type 1, 2,
or 3 of the RYR mRNA. None of 26 B cell lines from immature stages,
i.e., pro-B, common-B, and pre-B types, expressed the RYR (Table III). Many of the nonlymphocytic lines
(myelocytic, megakaryocytic, erythroid, and nonlymphocytic,
nonmyelocytic) expressed RYR mRNA, mainly type 3 (Tables I and II).
Expression of type 3 RYR mRNA was especially manifested in
megakaryocytic and nonlymphocytic, nonmyelocytic lines. Some of the
cell lines, such as U937, SupT1, and SKW6.4, expressed more than a
single isoform (Fig. 4).
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and other stimuli
It has been reported previously that TGF- treatment induced
expression of type 1 and type 3 RYR mRNA in HeLa cells and mink lung
epithelial cells, respectively (17, 23). We examined
whether any isoform of RYR mRNA was induced in PBMCs by treatment
with TGF- (Table IV). In
addition, the effects of activation by Abs to T or B cell receptors,
mitogens, or chemokines on expression of the three RYRs were examined
to gain some insight regarding association of the RYRs with immune
function (Table IV). TGF- (100 pg/ml) treatment for 24 h
induced RYR2 mRNA in PBMCs. Although activation of T cells by PHA (1
µg/ml) significantly increased type 1 mRNA, neither Con A (1 µg/ml)
nor combination of anti-CD3 plus PMA increased expression of any
isoform of RYR mRNA. Activation of B cells by the combination of goat
F(ab)2 anti-human IgM Ab plus PMA increased
expression of type 2 RYR mRNA. LPS (1 µg/ml) caused no significant
increase in expression of any isoform of RYR mRNA in PBMCs. Treatment
with chemokines, SDF-1 (500 ng/ml), MIP1 (10 ng/ml), and RANTES
(100 ng/ml) increased expression of RYR2 mRNA. Type 1 RYR mRNA was also
increased 24 h after SDF-1. SDF-1 (500 ng/ml) had no effect
on RYRs mRNA. Type 3 RYR mRNA was not detected in PBMCs after any of
the above treatments (Table IV).
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and chemokines on the three RYRs were also
examined using Jurkat T and U937 cells. Although expression of type 3
RYR in Jurkat T cells has been reported, the Jurkat T cells, which had
been purchased from ATCC and maintained in our laboratory, showed no
expression of any isoforms of RYR mRNA (Fig. 4). Stimulation with
SDF-1, MIP1, and TGF- induced expression of RYR2 and RYR3 mRNA
in the Jurkat T cells grown in our laboratory (Fig. 5). SDF-1 induced only RYR3 mRNA.
RANTES also induced RYR3 mRNA, but the degree of induction was smaller
than that by other chemokines (data not shown). Similar findings were
obtained using U937 cells, where the above agents caused enhancements
in expression of RYRs; the U937 cells expressed all three types of RYR
mRNA before stimulation (data not shown).
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Using the cell lines expressing RYR mRNA, we examined the effects
of the RYR-stimulating agents caffeine, 4CmC, and ryanodine on
Ca2+ levels. Caffeine (1–50 mM) dose-dependently
increased [Ca2+]i in
DAKIKI B cells and SupT1 cells. This increase was totally blocked by
the addition of excess extracellular EGTA (5 mM), indicating that
caffeine induces Ca2+ influx without eliciting
Ca2+ release from the internal
Ca2+ store. Caffeine (1–50 mM) caused neither
Ca2+ release nor Ca2+
influx in other cell lines. Within a range of 100 µM–1 mM, 4CmC
caused a dose-dependent increase in
[Ca2+]i in SupT1, DAKIKI,
SKW6.4, U937, and THP-1 cells (Fig. 6).
The 4CmC-induced increase in
[Ca2+]i in the cells was
not reduced by excess EGTA and hence involves mainly release from
internal stores. In Jurkat T cells, 4CmC (>1 mM) had no effect on
[Ca2+]i (Fig. 6A). In contrast, 4CmC (< 400 µM) caused
Ca2+ release after induction of RYR2 and RYR3 in
Jurkat T cells by treatment with SDF-1, MIP1 or TGF- (Fig. 6B). None of the above cell lines responded to ryanodine (1
µM–1 mM). There was no clearcut relationship between the kinetics of
Ca2+ changes and expression of isoforms. For
example, Ca2+ response to 4CmC in the
RYR1-expressing line THP-1 was more similar to that in Jurkat cells
treated with TGF- (RYR1 negative) than the
Ca2+ response seen in RYR1+
DAKIKI cells. The cell lines expressing all three isoforms, i.e.,
SKW6.4, U937, and SupT cells, tend to show relatively slow but
long-lasting increases in
[Ca2+]i in response to
4CmC (400 µM) compared with immediate but short-lasting increases in
[Ca2+]i seen in THP-1 or
Jurkat cells treated with TGF-. In addition to Jurkat T cells, we
examined H9 and HL-60 that gave negative RYR expression in RT-PCR
experiments. Neither cell line showed an increase in
[Ca2+]i in response to
4CmC at concentrations below 1 mM (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Type 3 RYR has recently been proposed to be responsible for a novel Ca2+ signaling pathway in T cells (10). This is based on pharmacological and molecular biological findings that include TCR-stimulated increases in cyclic ADP-ribose, cyclic ADP-ribose-mediated Ca2+ release, and expression of RYR3 (10). However, this hypothesis was drawn from observations made in Jurkat T cells, not primary T cells. Because only type 2 or 1 was expressed in primary peripheral T cells, RYR-mediated Ca2+ signaling in T cells must be examined based on genotypic and phenotypic expression of RYR isoforms in primary T cells. Our finding is not surprising in light of the RYR3-deficient animal model in which there is normal proliferation of T and B lymphocytes in response to mitogens or IL-2 (24).
Cloning of type 3 RYR gene has been made by two independent
laboratories (23, 25). Induction of type 3 RYR in mink
lung epithelial cells (Mv1Lu) by TGF- treatment led to cloning of
the RYR3 gene (previously named 4 gene) by one
of these laboratories (23). Similarly, induction of type 1
RYR has also been observed in HeLa cells, murine NIH3T3 fibroblasts,
and mammary epithelial cell line HC11 cells (17). Our RYR
induction study gave us some important insights as to potential
association of the RYRs with immune function. Although which cell
subsets responded to each treatment remains to be determined, we found
that type 1 and/or type 2 RYR mRNA were inducible by a variety of
treatments in PBMCs (Table IV). Substantial increases in RYR1 and RYR2
mRNA expression by PHA, cross-linking surface IgM, and chemokines
(SDF-1, MIP1, and RANTES) suggest functional requirement of RYRs for
T cell signaling, B cell receptor-mediated B cell activation and
chemotaxis of immune cells. In the immune system, TGF- antagonizes T
cell proliferation and macrophage activation and regulates Ig class
switching in B cells. Thus, induction of RYR2 mRNA by TGF- in PBMCs
may be involved in some of the actions of TGF-.
A clear association of RYR expression with Ca2+
channel function was obtained from the studies with Jurkat T cells. At
least two laboratories have reported expression of RYR3 mRNA in Jurkat
T cells (10, 11), whereas Bennett et al. (26)
found no expression. Consistent with the finding by the latter group,
Jurkat T cells, which have been obtained from ATCC and maintained in
our laboratory, showed no constitutive expression of any RYR isoforms.
Nonetheless, Jurkat T cells were able to express not only type 3 but
also type 2 RYR when stimulated with SDF-1, MIP1, and TGF-.
Therefore, we suggest that expression of RYR in Jurkat T cells may be
clone or culture condition dependent. We have tested the effects of the
RYR-stimulating agent 4CmC on Ca2+ response in
Jurkat T cells before and after induction of the RYRs. In Jurkat T
cells that showed no constitutive expression of any isoform of the RYR,
>1 mM 4CmC did not cause any increase in
[Ca2+]i (Fig. 6A). In contrast, 400 µM 4CmC induced a significant
increase in [Ca2+]i in
Jurkat T cells that expressed RYR2 and RYR3 after the treatment with
TGF- (Fig. 6B). The 4CmC-induced increases in
[Ca2+]i were due to
Ca2+ release as shown in the absence of
extracellular Ca2+. Jurkat T cells treated with
chemokines (SDF-1, SDF-1, MIP1, and RANTES) also showed
significant Ca2+ release in response to 400 µM
4CmC. Therefore, the appearance of 4CmC-induced
Ca2+ release indicated that Jurkat T cells
treated with these chemokines or TGF- indeed expressed functional
RYRs. Consistent with that finding, 4CmC also induced
Ca2+ release in the RYR-expressing cell lines,
U937, THP-1, SKW6.4, DAKIKI, and Sup T cells, suggesting that these
cell lines not only express mRNA but also a functional
Ca2+ release channel. Contrasting with 4CmC data,
the effects of caffeine and ryanodine on
[Ca2+]i are not easily
interpreted. Caffeine, a classic ryanodine receptor activator, did not
induce Ca2+ release from the internal
Ca2+ store in any of these cell lines at
concentrations of 1–50 mM. However, caffeine (>25 mM) caused
significant Ca2+ influx without inducing
Ca2+ release in DAKIKI and SupT1 cells. In
addition to the Ca2+ influx, it has been found
that caffeine (1–10 mM) suppressed IP3R- or
RYR-mediated Ca2+ release in primary B cells and
DAKIKI cells (27). Similarly, ryanodine did not induce
Ca2+ release in any of these cell lines. Findings
from others also indicate lack of stimulatory effects of caffeine and
ryanodine on Ca2+ release in many types of
nonexcitable cells (11, 26). Therefore, pharmacological
properties of caffeine and ryanodine on cytoplasmic
Ca2+ response must be further investigated for
hemopoietic cells and other nonexcitable cells.
Expression of RYR isoforms, especially RYR1 and 2 in primary T cells, B
cells, and monocytes, suggests that there are multiple
Ca2+ release mechanisms that control the highly
complex Ca2+ signaling in immune cells. In T
cells, B cells, and monocytes, it is generally suggested that receptor
stimulation causes Ca2+ release and opening of
the SOC channels. Recent evidence suggests that the SOC is physically
and functionally coupled with IP3Rs and also with
RYRs (28). Interestingly, gating of the SOC seems to be
significantly affected by the isoform of the coupling partner. An
electrophysiological study in excised patches from cells transfected
with the SOC candidate human Trp3 (29) indicated that the
human Trp3 channel was effectively gated by caffeine or cyclic
ADP-ribose in the presence of RYR1 or RYR3 whereas RYR2 failed to
reconstitute gating of the channel (28). Therefore, an
increase or decrease in the expression of RYR isoforms may regulate
receptor-mediated Ca2+ responses by altering both
Ca2+ release and SOC function. Induction of
specific RYR isoforms by mitogens, chemokines, and TGF- also
suggests that such an alteration in the RYR expression is required for
regulating intracellular Ca2+ responses during
certain processes of immune activation, such as chemotaxis of immune
cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Yoshitatsu Sei, Department of Anesthesiology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799. E-mail address: ysei{at}usuhs.mil
3 Abbreviations used in this paper: [Ca2+]i, intracellular free Ca2+ concentration; 4CmC, 4-chloro-m-cresol; IP3, inositol 1,4,5-trisphosphate; mIg, membrane Ig; RYR, ryanodine receptor; SOC, store-operated Ca2+ channel; SDF-1, stromal cell-derived factor 1; MIP1, macrophage-inflammatory protein-1.
Received for publication May 30, 2001. Accepted for publication August 23, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 L. Bracci, M. Vukcevic, G. Spagnoli, S. Ducreux, F. Zorzato, and S. Treves Ca2+ signaling through ryanodine receptor 1 enhances maturation and activation of human dendritic cells J. Cell Sci., July 1, 2007; 120(13): 2232 - 2240. [Abstract] [Full Text] [PDF]
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A. Guidarelli, L. Cerioni, and O. Cantoni Inhibition of complex III promotes loss of Ca2+ dependence for mitochondrial superoxide formation and permeability transition evoked by peroxynitrite J. Cell Sci., June 1, 2007; 120(11): 1908 - 1914. [Abstract] [Full Text] [PDF]
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 M. Stolk, M. Leon-Ponte, M. Merrill, G. P. Ahern, and P. J. O'Connell IP3Rs are sufficient for dendritic cell Ca2+ signaling in the absence of RyR1 J. Leukoc. Biol., September 1, 2006; 80(3): 651 - 658. [Abstract] [Full Text] [PDF]
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 G. Cherednichenko, A. V. Zima, W. Feng, S. Schaefer, L. A. Blatter, and I. N. Pessah NADH Oxidase Activity of Rat Cardiac Sarcoplasmic Reticulum Regulates Calcium-Induced Calcium Release Circ. Res., March 5, 2004; 94(4): 478 - 486. [Abstract] [Full Text] [PDF]
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B. Xiao, H. Masumiya, D. Jiang, R. Wang, Y. Sei, L. Zhang, T. Murayama, Y. Ogawa, F. A. Lai, T. Wagenknecht, et al. Isoform-dependent Formation of Heteromeric Ca2+ Release Channels (Ryanodine Receptors) J. Biol. Chem., October 25, 2002; 277(44): 41778 - 41785. [Abstract] [Full Text] [PDF]
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K. Paul-Pletzer, T. Yamamoto, M. B. Bhat, J. Ma, N. Ikemoto, L. S. Jimenez, H. Morimoto, P. G. Williams, and J. Parness Identification of a Dantrolene-binding Sequence on the Skeletal Muscle Ryanodine Receptor J. Biol. Chem., September 13, 2002; 277(38): 34918 - 34923. [Abstract] [Full Text] [PDF]
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