Expression of the Ryanodine Isoforms in Immune Cells Eiji Hosoi, Chiharu Nishizaki, Kathleen L. Gallagher, Hadley W. Wyre, Yoshinobu Matsuo and Yoshitatsu Sei This information is current as of September 23, 2021. J Immunol 2001; 167:4887-4894; ; doi: 10.4049/jimmunol.167.9.4887 http://www.jimmunol.org/content/167/9/4887 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Expression of the Isoforms in Immune Cells1

Eiji Hosoi,*† Chiharu Nishizaki,‡ Kathleen L. Gallagher,* Hadley W. Wyre,* Yoshinobu Matsuo,‡ and Yoshitatsu Sei2*

Ryanodine receptor (RYR) is a Ca2؉ channel that mediates Ca2؉ release from intracellular stores. We have used RT-PCR analysis and examined its expression in primary peripheral mononuclear cells (PBMCs) and in 164 hemopoietic cell lines. In PBMCs, type .RYR (RYR1) was expressed in CD19؉ B lymphocytes, but less frequently in CD3؉ T lymphocytes and in CD14؉ monocytes 1 Type 2 RYR (RYR2) was mainly detected in CD3؉ T cells. Induction of RYR1 and/or RYR2 mRNA was found after treatment with stromal cell-derived factor 1, macrophage-inflammatory -1␣ (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؉ Downloaded from 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 ␮M) 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 dem- onstrate 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. The Journal of Immunology, http://www.jimmunol.org 2001, 167: 4887–4894.

alcium ions play a critical role in the activation of the after surface receptor ligation (7, 8). Phospholipase C␥ is then immune cells that are responsible for cellular and hu- recruited to an upstream tyrosine kinase via its SH2 domains and C moral immunity (1–3). In the process of immune re- activated by phosphorylation. Phospholipase C␥ activation leads sponses, Ag binding to the surface receptor stimulates the immune to the hydrolysis of phosphatidylinositol 4,5-bisphosphate, yield- cells to eventually eliminate foreign Ags. Elevation of intracellular ing IP3 and diacyl glycerol. IP3 then mediates the activation of 2ϩ 2ϩ 3 2ϩ free Ca concentration ([Ca ]i) is an early and critical event in Ca release from stores in the endoplasmic reticulum through the by guest on September 23, 2021 the biochemical cascade of signal transduction pathways, which IP3 receptor. Therefore, calcium mobilization after receptor cross- include activating specific transcription factors (i.e., NF-␬B, JNK, linking in the immune cells has been explained almost solely by NF-AT, etc.), and thus in a variety of later events in the immune IP -mediated mechanisms. ϩ 3 cell activation (4). Therefore, the regulation of Ca2 signaling de- 2ϩ Although IP3 is a key messenger regulating [Ca ]i, recent stud- termines the ultimate response of an immune cell. ies have postulated the possibility that the ryanodine receptor An early manifestation of mitogen- or cell surface receptor- (RYR) contributes to the IP -insensitive component of Ca2ϩ sig- 2ϩ 3 stimulated immune cell activation is a biphasic increase in [Ca ]i naling in immune cells (9–12). The RYR was originally found in 2ϩ which is the result of rapid Ca release from intracellular stores the sarcoplasmic reticulum of (type 1 receptor; 2ϩ 2ϩ followed by sustained Ca influx through store-operated Ca RYR1) and cardiac muscle (type 2 receptor; RYR2) (13–15). Ca2ϩ 2ϩ channels (SOC) (5, 6). Ca release from intracellular stores is release from the sarcoplasmic reticulum through these receptors consequent to inositol 1,4,5-trisphosphate (IP3) formation. The ac- plays a central role in regulating the contraction of skeletal and tivation of multiple protein tyrosine kinases occurs immediately cardiac muscle fibers. A third type of RYR (type 3 receptor; RYR3) has been detected in specific regions of the brain, non- *Department of Anesthesiology, Uniformed Services University of the Health Sci- muscle tissues, and also skeletal muscle (16–18). We recently ences, Bethesda, MD 20814; †Department of Medical Technology, School of Medical demonstrated that human B cells express a RYR that is identical Sciences, University of Tokushima, Tokushima, Japan; and ‡Fujisaki Cell Center, Hayashibara Biochemical Laboratories, Inc., Okayama, Japan with skeletal muscle type I by RFLP studies and sequencing anal- Received for publication May 30, 2001. Accepted for publication August 23, 2001. ysis of partially cloned cDNA (12). In addition, 4-chloro-m-cresol (4CmC), a potent activator of the RYR (19), induced Ca2ϩ release The costs of publication of this article were defrayed in part by the payment of page 2ϩ charges. This article must therefore be hereby marked advertisement in accordance after depleting IP3-sensitive Ca pools in B cells (12). These with 18 U.S.C. Section 1734 solely to indicate this fact. results suggested that human B cells express functional RYR1 that 1 This work was supported by grants from the Uniformed Services University of the is involved in regulating Ca2ϩ signaling, perhaps in conjunction Health Sciences (R08078) and the Malignant Hyperthermia Association of the United States (G18090). 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 2 Address correspondence and reprint requests to Dr. Yoshitatsu Sei, Department of 3 Anesthesiology, Uniformed Services University of the Health Sciences, 4301 Jones cells (9). In both T cell lines, cyclic ADP-ribose increased [ H]ry- Bridge Road, Bethesda, MD 20814-4799. E-mail address: [email protected] anodine binding and induced Ca2ϩ release from intracellular Ca2ϩ 3 2ϩ 2ϩ Abbreviations used in this paper: [Ca ]i, intracellular free Ca concentration; stores (9, 10). The isoform of the RYR expressed in Jurkat T cells 4CmC, 4-chloro-m-cresol; IP3, inositol 1,4,5-trisphosphate; mIg, membrane Ig; RYR, ryanodine receptor; SOC, store-operated Ca2ϩ channel; SDF-1, stromal cell-derived was identified to be type 3 (10, 11). Therefore, the RYR3 has been ␣ ␣ 2ϩ factor 1; MIP1 , macrophage-inflammatory protein-1 . proposed to control [Ca ]i in response to cyclic ADP-ribose

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 4888 RYR ISOFORMS IN IMMUNE CELLS

during T cell activation (10). These findings of the RYR in T and Selective RT-PCR followed by RFLP analysis B cells allowed us to hypothesize that the RYRs are more widely 2ϩ Total RNA was extracted using the SV Total RNA Isolation System (Pro- expressed and responsible for regulating [Ca ]i in immune cells mega, Madison, WI), and reverse transcription was performed to the first than currently thought. strand of cDNA using a cDNA synthesis kit (Promega). Synthesized cDNA In this study, we have investigated expression of all three iso- was then amplified by RT-PCR using a primer set which selectively am- forms of the RYR in human primary T cells, B cells, and mono- plifies specific isoform of the RYR. Using the same downstream primer, 5Ј-dC-AGATGAAGCATTTGGTCTCCAT-3Ј, and an isoform-specific cytes using selective RT-PCR followed by RFLP analysis. We also upstream primer: JBR1, 5Ј-dG-ACATGGAAGGCTCAGCTGCT-3Ј; examined a total of 164 human hemopoietic cell lines (36 T cell, JBR2, 5Ј-dAAGGAGCTCCCCACGAGAAGT-3Ј; and JBR3, 5Ј-dAA 92 B cell, 19 myelomonocytic, 11 megakaryocytic, 3 erythrocytic, GAGGAAGAAGCGATGGT-3Ј,anϳ1200-bp product was recognized and 3 nonlymphocytic, nonmyelocytic) to determine the lineage from the 3Ј-regions of RYR1, RYR2, and RYR3, respectively. PCR am- plifications were conducted using the Expand Long PCR system (Boehr- and differentiation specificity of the expression of the 3 RYRs. The inger Mannheim, Indianapolis, IN). PCR was performed in a 50-␮l reaction possibility that any isoform of RYR is induced by stimulation with mixture containing 100 ng DNA, 15 pmol of each primer, 0.5 mM dNTPs, mitogens, chemokines, and other stimuli were investigated to gain 2.5 U Expand Long polymerase mixture and Expand Long PCR buffer 3 insight of the roles of this Ca2ϩ release channel in immune func- (Boehringer Mannheim). The PCR amplification conditions were 95°C for tion. Finally, to verify the functional expression of the RYRs, 2 min, followed by 40 cycles of 95°C for 1 min, 55°C for 2 min, and 68°C 2ϩ for 3 min, followed by a 7-min extension at 68°C. The RT-PCR products Ca release by RYR-stimulating agents was assessed using the were then digested with selected restriction enzymes, HgaI, BsmI, and cell lines expressing the RYR mRNA. A global view of RYR HindIII to identify the RYR isoform. Based on the known sequences of expression in human immune cells was addressed in this study. 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 Downloaded from RYR3. BsmI cuts only the 1083-bp RYR2 and produces 762- and 321-bp Materials and Methods fragments. HindIII cuts the 1015-bp RYR3 product to make 537- and Reagents 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 Stroma-derived factors 1␣ and 1␤ (SDF-1␣ and -1␤), macrophage-inflam- resolved by electrophoresis on a 2% agarose gel and visualized on a UV matory protein-1␣ (MIP1␣), TGF-␤, RANTES, nerve growth factor, and transilluminator. As a control of mRNA input, ␤-actin mRNA levels were 4CmC were obtained from Calbiochem (San Diego, CA). PHA, Con A determined for each sample in separate RT-PCR. For ␤-actin amplification, (type IV), LPS (Escherichia coli; B5W), PMA, and were from PCR was performed with 25 cycles to ensure that the amplification was http://www.jimmunol.org/ Sigma (St. Louis, MO). Fluo-3 acetoxymethyl ester was obtained from completed within the linear range. The sequences of primers for ␤-actin Molecular Probes (Eugene, OR). Anti-CD19 and anti-CD3 mAbs were were 5Ј-dAAGAGAGGCATCCTCACCCT-3Ј (sense) and 5Ј-dTGCT from BD PharMingen (San Diego, CA). Total RNA isolated from normal GATCCACATCTGCTGGA-3Ј (antisense). In some experiments, the sig- heart and mRNA from brain was purchased from Invitrogen nal ratio of RYR to ␤-actin was determined on the basis of the ratio of the (Carlsbad, CA). intensity of the PCR product compared with the corresponding ␤-actin band. The PCR products were imaged, and the relative OD of each band Primary cells, cell lines, and tissues was measured and analyzed using NIH Image software. Buffy coats were obtained from healthy blood donors at the National In- Ca2ϩ mobilization test using B cells stitutes of Health Blood Bank (Bethesda, MD). PBMCs were isolated by ϩ ϩ 2ϩ by guest on September 23, 2021 Ficoll-Hypaque density gradient centrifugation. CD3 T cells, CD19 B Relative changes in [Ca ]i were derived from changes in the fluorescence cells, and CD14ϩ monocytes were purified from the PBMCs using an intensity of fluo-3-loaded cells (21). Cells (2 ϫ 106/ml) were loaded with Ab-magnetic bead isolation system (Dynal, Oslo, Norway). Cells (107 1 ␮M fluo-3 acetoxymethyl ester in subdued light (30 min, 25°C). Cells cells) were first incubated with Dynabeads coated with anti-CD3 mAb for were then washed once with HBSS, resuspended in 1 ml of HBSS, and 30 min at 4°C. CD3ϩ cells attaching to the beads were isolated after three analyzed by FACScan (Becton Dickinson). Forward and right angle scatter washes with HBSS. Unattached cells were then incubated with anti-CD19 signals were displayed on a linear scale, with the forward scatter adjusted beads for 30 min at 4°C. CD19ϩ cells were isolated after three washes with to gate cells from debris. The fluo-3 fluorescence (excitation at 488 nm HBSS, and unattached cells were incubated with anti-CD14 beads for 30 with emission at 525 nm) was detected after separation with a 530 (FL-1) min at 4°C. CD14ϩ cells were then isolated after three washes with HBSS. band pass filter. FL-1 fluorescence was recorded, amplified, and displayed Jurkat, SupT1, H9, CEM, SKW6.4, DAKIKI, THP-1, and U937 were ob- tained 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 supple- mented with 10% FCS (HyClone, Logan, UT), 2 mM L-glutamine, 100 U penicillin, and 100 ␮g/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 homogeniz- ers in buffer containing 50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 10 ␮g/ml leupeptin, 10 ␮g/ml aprotinin, and 25 ␮g/ml p-nitrophenylguanidinoben- zoate and then incubated for 20 min at 4°C. After centrifugation at FIGURE 1. A, Agarose gel electrophoresis of PCR products after se- 14,000 ϫ g for 15 min, the supernatants were collected and analyzed for lective RT-PCR for three isoforms of RYR. cDNA obtained from skeletal total protein (BCA protein assay kit; Pierce, Rockford, IL). The protein (S.) muscle, cardiac (C.) muscle, and brain was amplified using primer sets ␮ samples (10–75 g/lane) were separated using SDS-PAGE on a 10% Tris- that specifically amplify type 1 (RYR1), type 2 (RYR2), or type 3 (RYR3) glycine gel. After separation, the were transferred to a polyvinyli- isoforms. B, Agarose gel electrophoresis of digested or undigested RT- dene difluoride membrane (Millipore, Bedford, MA), and then probed with monoclonal anti-RYR Abs (Affinity Bioreagents, Golden, CO). Alkaline PCR products with restriction enzymes. After RT-PCR, the amplified frag- phosphatase-conjugated monoclonal anti-rabbit IgG Ab (Sigma) was used ments of RYR1 (1112 bp from skeletal muscle), RYR2 (1083 bp from to detect the primary rabbit Abs. Chemiluminescence detection was per- cardiac muscle), and RYR3 (1015 bp from brain) were digested with HgaI, formed using the alkaline phosphatase substrate CSPD (Tropix, BsmI, or HindIII to confirm the specificity of the PCR product. M, Mo- Bedford, MA). lecular size marker. The Journal of Immunology 4889

FIGURE 2. Agarose gel electrophoresis of PCR products after selective RT-PCR for three isoforms of RYR. The PCR products from three different individuals (1, 2, and 3) are shown. cDNAs obtained from purified CD3ϩ T cells, CD19ϩ B cells, and CD14ϩ monocytes were amplified using primer sets that specifically amplify type 1 (RYR1), type 2 (RYR2), or type 3 (RYR3) isoform. The specificity of the PCR products was confirmed with RFLP as shown in Fig. 1. ␤-Actin was also amplified for each sample to

estimate levels of cDNA input from each sample. S., skeletal; C., cardiac; Downloaded from M, molecular size marker.

FIGURE 3. Western blot analysis of RYR. Skeletal muscle, 10 ␮g total protein from skeletal muscle tissue; PBMCs (PMCs), 75 ␮g protein from on a logarithmic scale. For each experiment, the fluo-3-loaded cells were human PBMCs before magnetic bead separation; CD3ϩ T cells, 75 ␮g analyzed to obtain an unstimulated baseline. Cells were then exposed to ϩ ϩ caffeine, ryanodine, or 4CmC and analyzed continuously at rates of 400- protein from purified CD3 T cells; CD19 B cells, 75 ␮g protein from ϩ ϩ ϩ ␮ 1000 cells/s. The percentage of fluo-3 cells (or mean fluorescence channel purified CD19 B cells; and CD14 monocytes, 75 g protein from pu- http://www.jimmunol.org/ of fluo-3) relative to unstimulated baseline was analyzed to monitor the rified CD14ϩ monocytes. Molecular size markers are not included in this 2ϩ magnitude of elevation in [Ca ]i in cells using CellQuest software (Bec- figure. Arrow, Position of the ryanodine receptor. Dark staining in the ton Dickinson). middle area of the blot is nonspecific staining. Results Expression of RYR isoforms in human primary mononuclear respectively (Refs. 15 and 17 and Fig. 1). As predicted based on cells previous observations, RYR1 mRNA was highly expressed in Expression of the three isoforms of RYR mRNA was investigated skeletal muscle. RYR3 mRNA was also detected at lower levels by selective RT-PCR followed by RFLP analysis. In this method, than RYR1 in skeletal muscle (Fig. 1A). In cardiac muscle, RYR2 by guest on September 23, 2021 cDNA for RYR is synthesized from mRNA by reverse transcrip- mRNA was dominantly expressed compared with types 1 and 3 tion and amplified by selective PCR using the isoform-specific (Fig. 1A). In brain, all three isoforms were highly expressed (Fig. primers. The isoform-specific PCR primers for amplification of the 1A). Specificity of the selective RT-PCR products was then exam- human RYR1, RYR2, and RYR3 were designed to produce an ined by RFLP analysis. The RT-PCR products amplified for the ϳ1200-bp product from the 3Ј-region of the RYR1, RYR2, and RYR1, RYR2, and RYR3 from skeletal muscle, cardiac muscle, RYR3. To further confirm specificity of the RT-PCR products, the and brain, respectively, were digested with restriction enzymes PCR products were digested with selected restriction enzymes HgaI, BsmI, and HindIII. The PCR product amplified for the HgaI, BsmI, and HindIII. Based on the sequences of human RYR1, RYR1 from skeletal muscle was cut into 692-, 349-, and 71-bp RYR2, and RYR3 available in the GenBank, HgaI, BsmI, and fragments by HgaI, but it was not digested with BsmIorHindIII HindIII cut at a unique site in the amplified sequences for RYR1, (Fig. 1B). The amplicon for RYR2 from cardiac muscle was cut RYR2, and RYR3, respectively. We tested our two-step method by into 762- and 321-bp fragments by BsmI (Fig. 1B). The product for examining cDNAs from skeletal muscle, cardiac muscle, and brain RYR3 from brain was cut into 537- and 478-bp fragments only by which have been shown to express type 1, type 2, and type 3, HindIII (Fig. 1B).

Table I. RYR expression in primary T and B cells and monocytes and cell lines

No. of Positive Individuals (or cell lines) (%) No. of Individuals (or Cell Types cell lines) Examined RYR1 RYR2 RYR3 RYRa 1, 2, and 3

Primary cells CD3ϩ T cells 9 1 (11.1) 4 (44.4) 0 CD19ϩ B cells 9 5 (55.5) 2 (22.2) 0 CD14ϩ monocytes 9 1 (11.1) 0 0 Cell lines T cell lines 36 2 (5.6) 3 (8.3) 2 (5.6) 1 (2.8)a B cell lines 92 1 (1.1) 8 (8.7) 9 (9.3) 1 (1.1)a Myelomonocytic lines 19 1 (5.3) 1 (5.3) 3 (15.8) 1 (5.3)a Megakaryocytic lines 11 0 0 6 (54.5) 0 Erythroid lines 3 0 1 (33.3) 1 (33.3) 0 Non-L, non-M cell lines 3 0 0 3 (100) 0

a Cell lines expressing multiple isoforms, SupT, SKW6.4, and U937. 4890 RYR ISOFORMS IN IMMUNE CELLS

Using this selective RT-PCR/RFLP method, we examined the from the donors that we examined. We performed SDS-PAGE expression of three isoforms of RYR mRNA in T cell, B cell, and immunoblot analysis to examine the protein expression in mono- monocyte populations purified from nine different donors (Fig. 2 nuclear cells from the donor who showed high levels of RYR1 and Table I). Type 1 RYR was detected in freshly isolated CD19ϩ mRNA in B cells and monocytes. The immunoblot with monoclo- B lymphocytes from five of nine donors. The RYR1 mRNA was nal anti-RYR Ab (clone 34-C) revealed the presence of the im- less frequently detected in CD3ϩ T lymphocytes and CD14ϩ munoreactive protein that was similar in size to the protein from monocytes. Type 2 RYR was mainly detected in CD3ϩ T cells. For skeletal muscle in purified CD19ϩ B cells and CD14ϩ monocytes type 3 amplification, we frequently observed smear without a dis- (Fig. 3). Some immunoreactivity was seen in purified CD3ϩ T cell tinguishable single band of PCR product, whereas RYR3 mRNA and total mononuclear cell preparation (Fig. 3). from control brain tissue was successfully amplified in every ex- periment (Fig. 2). The smearing was not due to overexpression of Expression of RYR isoforms in human hemopoietic cell lines type 3 mRNA, because the product was never detected even in A large array of human hemopoietic cell lines at various stages of samples diluted up to 104 times. Because ␤-actin was highly ex- differentiation allows us to determine the lineage and differentiation pressed in all cDNA samples applied for the RYR3 amplification, specificity of the expression of and proteins. To investigate we concluded that type 3 RYR mRNA was not present at detect- specificity of expression of RYR isoforms, we examined total 164 able levels in peripheral T cell, B cell, and monocyte populations human cell lines, consisting 36 T and 92 B cell leukemia lines and 19 Downloaded from Table II. RYR expression in human nonlymphocytic and T cell leukemia cell linesa

Cell Line Originb RYR Cell Line Origin RYR

Myeloid T-Blast-I MR-87 AUL Ϫ MOLT-10 ALL Ϫ KG-1 AML Ϫ T-Blast-II ML1 AML Ϫ RPMI-8402 ALL Ϫ http://www.jimmunol.org/ HL-60 APL Ϫ CCRF-CEM ALL Ϫ PL-21 CML Ϫ DND-41 ALL Ϫ KCL-22 CML Ϫ HPB-ALL ALL Ϫ GDM-1 CML Ϫ HD-MAR-2 HD? Ϫ KU-812 CML 2ϩ STEER-92 LY Ϫ SCC3 AML 3ϩ/Ϫ MOLT-13 ALL Ϫ HIG CML 3ϩϩ ALL-SIL ALL Ϫ SKH-1 CML Ϫ T-Blast-III HOR CML Ϫ SUPT1 LY 1,2,3ϩ Ϫ Ϫ

EOL-1 EOL TALL-1 ALL by guest on September 23, 2021 Monocytoid MOLT-3/4 ALL Ϫ SUM90-7 AMMOL 3ϩ P12/Ichikawa ALL Ϫ P31/Fujioka AMOL 3ϩ/Ϫ KOPT-K1 ALL Ϫ IMS-M1 AMOL Ϫ MOLT-16 ALL 3ϩ/Ϫ MOLM-13 AMOL Ϫ Jurkat LY 2,3** THP-1 AMOL 1ϩϩ PF-382 ALL Ϫ U937 LY 1,2,3ϩϩ HAY-92N LY Ϫ Megakaryocytic HSB-2 ALL 2ϩ MEG-01 CML 3ϩ Willow-89 ALL Ϫ TS9;22 CML 3ϩ PEER ALL Ϫ YS9;22 CML Ϫ T-Blast-IV SS9;22 CML 3ϩ SKW-3 CLL Ϫ JURL-MK1 CML 3ϩ/Ϫ A3/Kawakami ALL 3ϩ/Ϫ JURL-MK2 CML Ϫ MAT ALL Ϫ M-07E MEGL Ϫ KARPAS-299 LY 2ϩϩ UT-7 MEGL 3ϩϩ T-Blast-V MOLM-1 CML Ϫ HUT-102 MF Ϫ MOLM-7 CML Ϫ C5/MJ NT Ϫ CMK-86 MEGL 3ϩ/Ϫ MT-4 NT Ϫ Erythroid MT-2 NT 1ϩ K562 CML 2ϩ ED-S ATL 2ϩ HEL EL 3ϩ/Ϫ H9 ALL Ϫ KMOE EL Ϫ SALT-3 ATL Ϫ Non-L, non-M IZ-86 ATL 1ϩ SU-DHL-1 LY 3ϩ MT-1 ATL Ϫ HDLM-2 HD 3ϩ HUT-78 SS Ϫ L428 HD 3ϩ

a Classification and definition of T cell lines (22): T-Blast-I, HLA-DRϩ, CD10ϩ, TdTϩ, and T-Agϩ (nearly identical with CD3); T-Blast-II, HLA-DRϪ, CD10ϩ, TdTϩ, and T-Agϩ; T-Blast-III, HLA-DRϪ, CD10Ϫ, TdTϩ, and T-Agϩ; T-Blast-IV, HLA-DRϪ, CD10Ϫ, TdTϪ, and T-Agϩ; T-Blast-V, HLA-DRϩ, CD10Ϫ, TdTϪ, T-Agϩ, and infected with human T cell leu- kemia virus-1. RYR 1, 2 and 3, expression of RYR isoforms, type 1, 2, or 3; ϩϩ, high level of expression; ϩ, moderate level expressed when stimulated with chemokines such as ,ءء ;of expression; ϩ/Ϫ, marginal expression; Ϫ, no detectable expression SDF-1␤. b AUL, acute undifferentiated leukemia; ALL, acute lymphoblastic leukemia; AML, acute myeloblastic leukemia; AMOL, acute monocytic leukemia; APL, acute promyelocytic leukemia; AMMOL, acute myelomonocytic leukemia; ATL, adult T cell leukemia; CML, chronic myelogenous leukemia; EL, erythroleukemia; EOL, eosinophilic leukemia; HD, Hodgkin’s disease; HCL, hairy cell leukemia; LY, lymphoma; MEGL, megakaryoblastic leukemia; MF, mycosis fungoides; NKL, natural killer leukemia; NT, normal retrovirus infected; SS, Se´zary syndrome. The Journal of Immunology 4891 myelomonocytic, 11 megakaryocytic, 3 erythrocytic, and 3 nonlym- mRNA, mainly type 3 (Tables I and II). Expression of type 3 RYR phocytic, nonmyelocytic cell lines (Tables I–III). In T cell lines, 22% mRNA was especially manifested in megakaryocytic and nonlym- of cell lines expressed type 1, 2, or 3 of the RYR mRNA. All T cell phocytic, nonmyelocytic lines. Some of the cell lines, such as U937, lines expressing detectable levels of RYR mRNA were from either SupT1, and SKW6.4, expressed more than a single isoform (Fig. 4). 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 Induction of RYR isoforms in primary mononuclear cells and ␤ than cells in blast-I and -II (22). Nine cells classified in blast-I or cell lines by TGF- and other stimuli blast-II did not express RYR mRNA. Similarly, 21% of B cell lines It has been reported previously that TGF-␤ treatment induced ex- (mainly mature B cell and plasma cell types) expressed type 1, 2, or pression of type 1 and type 3 RYR mRNA in HeLa cells and mink 3 of the RYR mRNA. None of 26 B cell lines from immature stages, lung epithelial cells, respectively (17, 23). We examined whether i.e., pro-B, common-B, and pre-B types, expressed the RYR (Table any isoform of RYR mRNA was induced in PBMCs by treatment III). Many of the nonlymphocytic lines (myelocytic, megakaryocytic, with TGF-␤ (Table IV). In addition, the effects of activation by Abs erythroid, and nonlymphocytic, nonmyelocytic) expressed RYR to T or B cell receptors, mitogens, or chemokines on expression of the

Table III. RYR expression in human B cell linesa

Cell Line Originb RYR Cell Line Origin RYR Downloaded from Pro-B cell SL-1 BL 2ϩ NALM-19 ALL Ϫ ABL-2 BL 2ϩ Common-B cell BALM-1/2 ALL Ϫ NALM-20 ALL Ϫ BALM6-8 ALL Ϫ NALM-21 ALL Ϫ MIDDLE-91 LY Ϫ NALM-24 ALL Ϫ U-698-M LY Ϫ Ϫ Ϫ

NALM-29 AUL Tanaka LY http://www.jimmunol.org/ HAL-01 ALL Ϫ MANCA-2 LY Ϫ OM9;22 ALL Ϫ JOK-1 HCL 3ϩ/Ϫ BV-173 ALL Ϫ SKW-4 LY 2ϩ/Ϫ KID-92 ALL Ϫ BALM3-5 LY Ϫ REH ALL Ϫ SU-DHL-4 LY Ϫ NALL-1 ALL Ϫ MLB-1084 LY Ϫ NALM-16 ALL Ϫ BAL-KH ALL Ϫ KOPN-K ALL Ϫ BOAR-88 LY 2ϩ NALM-33 ALL Ϫ BALM-9 BL 2ϩ PRE-B cell BALM-13 BL Ϫ NALM-1 CML Ϫ BALM-16 ALL Ϫ by guest on September 23, 2021 NALM6-13 ALL Ϫ BALM-18 ALL Ϫ NALM-17 ALL Ϫ BALM-19 ALL Ϫ NALM-26 ALL Ϫ BALM-24 ALL Ϫ INC ALL Ϫ BALM-25 ALL Ϫ LAZ-221 ALL Ϫ BALM-26 ALL Ϫ KOPN-8 ALL Ϫ Sc-1 LY Ϫ TAHR-87 ALL Ϫ Ri-1 LY Ϫ P30/Ohkubo ALL Ϫ BALL-1 ALL Ϫ KLM-2 AMOL Ϫ HAIR-M HCL Ϫ UDD-OCT ALL Ϫ JC-1 HCL Ϫ UDD-88 ALL Ϫ Plasma cell B cell AM01 MM Ϫ TREE-92 ALL Ϫ RPMI-8226 MM Ϫ BAY-91 ALL Ϫ U-266 MM Ϫ Black-93A BL Ϫ ARH-77 MM Ϫ EB-3 BL Ϫ L-363 MM 3ϩ RAJI BL Ϫ OPM-2 MM Ϫ HR1K BL Ϫ MOLP-2 MM Ϫ B35M BL Ϫ KM-1 MM Ϫ DAUDI BL 2ϩϩ KM-4 MM 3ϩ Namalwa BL Ϫ KM-5 MM Ϫ Ramos BL Ϫ KM-6 MM Ϫ BJAB BL Ϫ KM-7 MM 3ϩ DG-75 BL Ϫ KM-11 MM 3ϩ Chevallier BL Ϫ ILKM-10 MM 3ϩ DND-39 BL Ϫ IL-KM-12 MM 3ϩ/Ϫ NK-9 BL Ϫ KMS-18 MM Ϫ B46M BL Ϫ KHM-4 MM 3ϩ OGUN BL 3ϩ/Ϫ MOLP-5 MM 2ϩ AL-1 BL 2ϩ DAKIKI EBV 1ϩ SKW6.4 BL 1,2,3ϩ

a RYR 1, 2 and 3, expression of RYR isoform type 1, 2, or 3; ϩϩ, high level of expression; ϩ, moderate level of expression; ϩ/Ϫ, marginal expression; Ϫ, no detectable expression. b ALL, acute lymphoblastic leukemia; AUL, acute undifferentiated leukemia; AMOL, acute monocytic leukemia; CML, chronic myelogenous leukemia; BL, Burkitt’s lymphoma; EBV, EBV transformed; HCL, hairy cell leukemia; LY, lymphoma; MM, multiple myeloma. 4892 RYR ISOFORMS IN IMMUNE CELLS

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).

2ϩ Effects of RYR-stimulating agents on [Ca ]i using SupT1, Jurkat, DAKIKI, SKW6.4, U937, and THP-1 cell lines Using the cell lines expressing RYR mRNA, we examined the effects of the RYR-stimulating agents caffeine, 4CmC, and ryan- odine on Ca2ϩ levels. Caffeine (1–50 mM) dose-dependently in- 2ϩ creased [Ca ]i in DAKIKI B cells and SupT1 cells. This increase FIGURE 4. Expression of the RYR isoforms in immune cell lines. was totally blocked by the addition of excess extracellular EGTA cDNA obtained from CEM, SupT1, Jurkat, DAKIKI, SKW 6.4, THP-1, (5 mM), indicating that caffeine induces Ca2ϩ influx without elic- and U937 cells was amplified using primer sets which specifically amplify ϩ ϩ iting Ca2 release from the internal Ca2 store. Caffeine (1–50 type 1 (RYR1), type 2 (RYR2), or type 3 (RYR3) isoforms. The amplified 2ϩ 2ϩ products were digested with HgaI, BsmI, or HindIII to confirm the speci- mM) caused neither Ca release nor Ca influx in other cell ␮ Downloaded from ficity of the PCR products for all cell lines (not shown). M, molecular size lines. Within a range of 100 M–1 mM, 4CmC caused a dose- 2ϩ marker. dependent increase in [Ca ]i in SupT1, DAKIKI, SKW6.4, U937, 2ϩ and THP-1 cells (Fig. 6). The 4CmC-induced increase in [Ca ]i in the cells was not reduced by excess EGTA and hence involves three RYRs were examined to gain some insight regarding association mainly release from internal stores. In Jurkat T cells, 4CmC (Ͼ1 2ϩ Ͻ of the RYRs with immune function (Table IV). TGF-␤ (100 pg/ml) mM) had no effect on [Ca ]i (Fig. 6A). In contrast, 4CmC ( 400 ϩ

2 http://www.jimmunol.org/ treatment for 24 h induced RYR2 mRNA in PBMCs. Although ac- ␮M) caused Ca release after induction of RYR2 and RYR3 in tivation of T cells by PHA (1 ␮g/ml) significantly increased type 1 Jurkat T cells by treatment with SDF-1␤, MIP1␣ or TGF-␤ (Fig. mRNA, neither Con A (1 ␮g/ml) nor combination of anti-CD3 plus 6B). None of the above cell lines responded to ryanodine (1 ␮M–1 PMA increased expression of any isoform of RYR mRNA. Activa- mM). There was no clearcut relationship between the kinetics of Ca2ϩ changes and expression of isoforms. For example, Ca2ϩ re- tion 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 ␮g/ sponse to 4CmC in the RYR1-expressing line THP-1 was more ml) caused no significant increase in expression of any isoform of similar to that in Jurkat cells treated with TGF-␤ (RYR1 negative) 2ϩ ϩ RYR mRNA in PBMCs. Treatment with chemokines, SDF-1␣ (500 than the Ca response seen in RYR1 DAKIKI cells. The cell ␣

ng/ml), MIP1 (10 ng/ml), and RANTES (100 ng/ml) increased ex- by guest on September 23, 2021 pression 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). The effects of TGF-␤ 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). Stim- ulation with SDF-1␤, MIP1␣, and TGF-␤ induced expression of

Table IV. Induction of RYR 1, 2, and 3 mRNA in PMCsa

Treatment RYR1 RYR2 RYR3

Chemokines SDF-1␣, 0.5 ␮g/ml ϩϩϪ SDF-1␤, 0.5 ␮g/ml ϪϪϪ MIP-1␣, 10 ng/ml ϪϩϪ RANTES, 100 ng/ml ϪϩϪ Growth factors TGF-␤, 100 pg/ml ϪϩϪ FIGURE 5. Induction of RYR2 and RYR3 mRNA in Jurkat T cells in ␮ ϪϪϪ NGF, 1 g/ml response to chemokines and TGF-␤. Semiquantitative RT-PCR for the Mitogens ␣ ␮ ϪϩϪ RYR isoforms was performed. Jurkat T cells were stimulated with SDF-1 Con A, 1 g/ml ␤ ␤ ␣ PHA, 1 ␮g/ml ϩϩϪ (500 ng/ml), SDF-1 (500 ng/ml), TGF- (100 pg/ml), or MIP1 (10 ␤ LPS, 1 ␮g/ml ϪϪϪ ng/ml) for 24 h. -Actin expression was used as a control. Data are from Cross-linking receptors an experiment representative of two to three independent experiments. A, Anti-CD3 Ab ϩ PMA, 1 ng/ml ϪϪϪ Agarose gel electrophoresis of PCR products after selective RT-PCR for Anti-IgM Ab ϩ PMA, 1 ng/ml ϪϩϪ RYR1, RYR2, RYR3, and ␤-actin. B, Relative expression of RYR2 and a ϩ Ͼ RYR3 mRNA obtained by densitometric quantification of the RYR mRNA , Induction of the transcript; 1.5-fold increase was seen in two independent ␤ experiments; Ϫ, no detectable induction. Subjects who provided PMCs in this study transcript over the -actin mRNA transcript. S., skeletal; C., cardiac; M, were unrelated to those presented in Table I or Fig. 2. NGF, Nerve growth factor. molecular size marker. The Journal of Immunology 4893

DAKIKI B cell line as a type 1 RYR-positive line to model RYR1- mediated Ca2ϩ signaling in primary B cells (12). However, in the present study, we have found that only DAKIKI B cells expressed the RYR1 among 92 B cell lines. Thus, type 1 RYR mRNA, which is preferentially expressed in primary CD19ϩ B cells, seems to be down-regulated in other B cell lines. Conversely, type 3 RYR mRNA, which was not at all detected in PBMCs, has been detected in 4 T cell lines and 9 B cell lines. Interestingly, seven of these nine B cell lines were of multiple myeloma origin. Expression of type 3 was also robust in nonlymphocytic cell lines. Although we examined only two individuals, we did not find type 3 in either primary granule cell or platelet preparations (data not shown). Therefore, a shift of the RYR isoform from one type to another may be a noteworthy phenomenon found in hemopoietic cell lines. It is possible that this phenomenon is associated with oncogenesis or transformation of hemopoietic cells. Type 3 RYR has recently been proposed to be responsible for a novel Ca2ϩ signaling pathway in T cells (10). This is based on Downloaded from pharmacological and molecular biological findings that include TCR-stimulated increases in cyclic ADP-ribose, cyclic ADP-ri- bose-mediated Ca2ϩ release, and expression of RYR3 (10). How- ever, this hypothesis was drawn from observations made in Jurkat T cells, not primary T cells. Because only type 2 or 1 was ex- pressed in primary peripheral T cells, RYR-mediated Ca2ϩ signal- ing in T cells must be examined based on genotypic and pheno- http://www.jimmunol.org/ typic 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). FIGURE 6. Ca2ϩ release by 4CmC in immune cell lines. Cells were Cloning of type 3 RYR has been made by two independent incubated in Ca2ϩ-free (5 mM EGTA-containing) medium and stimulated laboratories (23, 25). Induction of type 3 RYR in mink lung epithelial ␮ 2ϩ ␤ with 4CmC (400 M). Changes in [Ca ]i after 4CmC were monitored cells (Mv1Lu) by TGF- treatment led to cloning of the RYR3 gene continuously for 10 min by measuring fluo-3 fluorescence using a FACS- (previously named ␤4 gene) by one of these laboratories (23). Simi- by guest on September 23, 2021 can as described in Materials and Methods. Graphs are linear density plots larly, induction of type 1 RYR has also been observed in HeLa cells, of fluo-3 fluorescence over time. A, Jurkat T cells. B, Jurkat T cells treated murine NIH3T3 fibroblasts, and mammary epithelial cell line HC11 ␤ with TGF- (100 pg/ml) for 24 h. Similar positive responses to 4CmC (400 cells (17). Our RYR induction study gave us some important insights ␮M) were observed in Jurkat T cells treated with SDF-1␣ and MIP1␣ (not as to potential association of the RYRs with immune function. Al- shown). C, SKW 6.4 B cells. D, DAKIKI B cells. E, U937 monocytic cells. F, THP-1 monocytic cells. G, SupT1 cells. though 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 lines expressing all three isoforms, i.e., SKW6.4, U937, and SupT increases in RYR1 and RYR2 mRNA expression by PHA, cross- ␣ cells, tend to show relatively slow but long-lasting increases in linking surface IgM, and chemokines (SDF-1, MIP1 , and RANTES) 2ϩ ␮ suggest functional requirement of RYRs for T cell signaling, B cell [Ca ]i in response to 4CmC (400 M) compared with immediate 2ϩ receptor-mediated B cell activation and chemotaxis of immune cells. but short-lasting increases in [Ca ]i seen in THP-1 or Jurkat cells treated with TGF-␤. In addition to Jurkat T cells, we examined H9 In the immune system, TGF-␤ antagonizes T cell proliferation and and HL-60 that gave negative RYR expression in RT-PCR exper- macrophage activation and regulates Ig class switching in B cells. 2ϩ Thus, induction of RYR2 mRNA by TGF-␤ in PBMCs may be in- iments. Neither cell line showed an increase in [Ca ]i in response to 4CmC at concentrations below 1 mM (data not shown). volved in some of the actions of TGF-␤. A clear association of RYR expression with Ca2ϩ channel func- Discussion tion was obtained from the studies with Jurkat T cells. At least two In this study, we have investigated expression of three isoforms of laboratories have reported expression of RYR3 mRNA in Jurkat T the RYR, RYR1, RYR2, and RYR3, in human primary T and B cells (10, 11), whereas Bennett et al. (26) found no expression. cells and monocytes and a total of 167 hemopoietic cell lines using Consistent with the finding by the latter group, Jurkat T cells, RT-PCR. In primary mononuclear cells, the isoform of RYR which have been obtained from ATCC and maintained in our lab- mRNA expressed in CD3ϩ T cell preparations was either type 1 or oratory, showed no constitutive expression of any RYR isoforms. type 2. Type 1 RYR was preferentially expressed in CD19ϩ B Nonetheless, Jurkat T cells were able to express not only type 3 but cells, but RYR2 mRNA was also detected in some individuals. also type 2 RYR when stimulated with SDF-1␤, MIP1␣, and RYR3 was not detected in T cell, B cell or monocyte preparations TGF-␤. Therefore, we suggest that expression of RYR in Jurkat T under the conditions we used. The expression pattern of the iso- cells may be clone or culture condition dependent. We have tested forms in cell lines was quite different from that seen in primary the effects of the RYR-stimulating agent 4CmC on Ca2ϩ response cells. Approximately 25% of T or B cell leukemia lines, mostly in Jurkat T cells before and after induction of the RYRs. In Jurkat mature cell types, expressed RYR1, RYR2, or RYR3 mRNA. T cells that showed no constitutive expression of any isoform of Ͼ 2ϩ Through previous screening of B cell lines, we have selected the the RYR, 1 mM 4CmC did not cause any increase in [Ca ]i 4894 RYR ISOFORMS IN IMMUNE CELLS

(Fig. 6A). In contrast, 400 ␮M 4CmC induced a significant in- 3. Ledbetter, J. A., L. E. Gentry, C. H. June, P. S. Rabinovitch, and A. F. Purchio. crease in [Ca2ϩ] in Jurkat T cells that expressed RYR2 and RYR3 1987. Stimulation of T cells through the CD3/T-cell receptor complex: role of i cytoplasmic calcium, protein kinase C translocation, and phosphorylation of after the treatment with TGF-␤ (Fig. 6B). The 4CmC-induced in- pp60c-src in the activation pathway. Mol. Cell Biol. 7:650. creases in [Ca2ϩ] were due to Ca2ϩ release as shown in the ab- 4. Dolmetsch, R. E., R. S. Lewis, C. C. Goodnow, and J. I. Healy. 1997. Differential i 2ϩ 2ϩ activation of transcription factors induced by Ca response amplitude and du- sence of extracellular Ca . Jurkat T cells treated with chemokines ration. [Published erratum appears in 1997 Nature 388:308.] Nature 386:855. (SDF-1␤, SDF-1␣, MIP1␣, and RANTES) also showed significant 5. Scharenberg, A. M., and J.-P. Kinet. 1998. 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Ryanodine receptor- not only express mRNA but also a functional Ca release chan- ankyrin interaction regulates internal Ca2ϩ release in mouse T-lymphoma cells. nel. Contrasting with 4CmC data, the effects of caffeine and ryan- J. Biol. Chem. 270:17917. 2ϩ 10. Guse, A. H., C. P. da Silva, I. Berg, A. L. Skapenko, K. Weber, P. Heyer, odine on [Ca ]i are not easily interpreted. Caffeine, a classic ry- 2ϩ M. Hohenegger, G. A. Ashamu, H. Schulze-Koops, B. V. Potter, and anodine receptor activator, did not induce Ca release from the G. W. Mayr. 1999. Regulation of calcium signalling in T lymphocytes by the ϩ internal Ca2 store in any of these cell lines at concentrations of second messenger cyclic ADP-ribose. Nature 398:70. 1–50 mM. However, caffeine (Ͼ25 mM) caused significant Ca2ϩ 11. Hakamata, Y., S. Nishimura, J. Nakai, Y. Nakashima, T. Kita, and K. Imoto. 1994. Involvement of the brain type of ryanodine receptor in T-cell proliferation. 2ϩ Downloaded from influx without inducing Ca release in DAKIKI and SupT1 cells. FEBS Lett. 352:206. In addition to the Ca2ϩ influx, it has been found that caffeine (1–10 12. Sei, Y., K. L. Gallagher, and A. S. Basile. 1999. Skeletal muscle type ryanodine 2ϩ receptor is involved in calcium signaling in human B lymphocytes. J. Biol. Chem. mM) suppressed IP3R- or RYR-mediated Ca release in primary 274:5995. B cells and DAKIKI cells (27). Similarly, ryanodine did not induce 13. Otsu, K., H. F. Willard, V. K. Khanna, F. Zorzato, N. M. Green, and Ca2ϩ release in any of these cell lines. Findings from others also D. H. MacLennan. 1990. Molecular cloning of cDNA encoding the Ca release channel (ryanodine receptor) of rabbit cardiac muscle sarcoplasmic reticulum. indicate lack of stimulatory effects of caffeine and ryanodine on J. Biol. Chem. 265:13472. 2ϩ Ca release in many types of nonexcitable cells (11, 26). There- 14. Takeshima, H., S. Nishimura, T. Matsumoto, H. Ishida, K. Kangawa, http://www.jimmunol.org/ fore, pharmacological properties of caffeine and ryanodine on cy- N. Minamino, H. Matsuo, M. Ueda, M. Hanaoka, T. Hirose, and S. Numa. 1989. Primary structure and expression from complementary DNA of skeletal muscle 2ϩ toplasmic Ca response must be further investigated for hemo- ryanodine receptor. Nature 339:439. poietic cells and other nonexcitable cells. 15. Zorzato, F., J. Fujii, K. Otsu, M. Phillips, N. M. Green, F. A. Lai, G. Meissner, and D. H. MacLennan. 1990. Molecular cloning of cDNA encoding human and Expression of RYR isoforms, especially RYR1 and 2 in primary rabbit forms of the Ca2ϩ release channel (ryanodine receptor) of skeletal muscle T cells, B cells, and monocytes, suggests that there are multiple sarcoplasmic reticulum. J. Biol. Chem. 265:2244. Ca2ϩ release mechanisms that control the highly complex Ca2ϩ 16. Ledbetter, M. W., J. K. Preiner, C. F. Louis, and J. R. 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