Effect of ryanodine receptor mutations on interleukin-6 release and intracellular calcium homeostasis in human myotubes from malignant hyperthermia-susceptible individuals and patients affected by central core disease

In this study we report for the first time the functional properties of human myotubes isolated from patients harboring the native RYR1 I4898T and R4893W mutations linked to central core disease. We examined two aspects of myotube physiology, namely excitation-contraction and excitation-secretion coupling. Our results show that upon activation of the ryanodine receptor (RYR), myotubes release interleukin-6 (IL-6); this was dependent on de novo protein synthesis and could be blocked by dantrolene and cyclosporine. Myotubes from the two patients affected by central core disease showed a 4-fold increase in the release of the inflammatory cytokine IL-6, compared with cells derived from control or malignant hyperthermia susceptible individuals. All tested myotubes released calcium from intracellular stores upon stimulation via surface membrane depolarization or direct RYR activation by 4-chloro-m-cresol. The functional impact on calcium release of RYR1 mutations linked to central core disease or malignant hyperthermia is different: human myotubes carrying the malignant hyperthermia-linked RYR1 mutation V2168M had a shift in their sensitivity to the RYR agonist 4-chloro-m-cresol to lower concentrations, whereas human myotubes harboring C-terminal mutations linked to central core disease exhibited reduced [Ca2+]i increase in response to 4-chloro-m-cresol, caffeine, and KCl. Taken together, these results suggest that abnormal release of calcium via mutated RYR enhances the production of the inflammatory cytokine IL-6, which may in turn affect signaling pathways responsible for the trophic status of muscle fibers.     

B-lymphocytes from malignant hyperthermia-susceptible patients have an increased sensitivity to skeletal muscle ryanodine receptor activators.

Malignant hyperthermia (MH) is a pharmacogenetic disease triggered by volatile anesthetics and succinylcholine in genetically predisposed individuals. The underlying feature of MH is a hypersensitivity of the calcium release machinery of the sarcoplasmic reticulum, and in many cases this is a result of point mutations in the skeletal muscle ryanodine receptor calcium release channel (RYR1). RYR1 is mainly expressed in skeletal muscle, but a recent report demonstrated the existence of this isoform in human B-lymphocytes. As B-cells can produce a number of cytokines, including endogenous pyrogens, we investigated whether some of the symptoms seen during MH could be related to the involvement of the immune system. Our results show that (i) Epstein-Barr virus-immortalized B-cells from MH-susceptible individuals carrying the V2168M RYR1 gene mutation were more sensitive to the RYR activator 4-chloro-m-cresol and (ii) their peripheral blood leukocytes produce more interleukin (IL)-1beta after treatment with the RYR activators caffeine and 4-chloro-m-cresol, compared with cells from healthy controls. Our result demonstrate that RYR1-mediated calcium signaling is involved in release of IL-1beta from B-lymphocytes and suggest that some of the symptoms seen during an MH episode may be due to IL-1beta production.     

The EGFP/hERG fusion protein alter the electrophysiological properties of hERG channels in HEK293 cells

EGFP (enhanced green fluorescent protein) tagged to either the N (amino)-terminus [EGFP/hERG (human ether-a-go-go-related gene)] or C (carboxyl)-terminus (hERG/EGFP) of hERG channel is used to study mutant channel protein trafficking for several years. However, it has been reported that the process can alter hERG channel properties. The aim of the study was to determine whether EGFP tagged to N-terminus of hERG channels would alter the cellular localizations and the electrophysiological properties of hERG channels compared with untagged hERG channels. The hERG channels tagged with or without EGFP were transiently expressed in HEK (human embryonic kidney) 293 cells using a lipofectamine method. HEK 293 cells expressing pCDNA3-hERG or pEGFP-hERG were double immunolabelled with anti-hERG and anti-calnexin (an ER marker protein) followed with FITC- and TRITC (tetramethylrhodamine β-isothiocyanate)-labelled secondary antibodies, respectively. Confocal laser scanning microscope was used to observe the cellular localization of EGFP-tagged hERG channels and untagged hERG channels. Patch-clamp technique was used to record whole cell currents. We found that the EGFP/hERG fusion protein and untagged hERG channels were both expressed not only on the cell surface membrane but also in the cytoplasm of HEK293 cells. The EGFP/hERG appeared to influence the hERG channel gating properties, including reduction of the peak tail current density, more rapid inactivation process, faster recovery from inactivation and faster deactivation kinetics compared with untagged hERG channels. Our results suggest that the EGFP/hERG channel alter the electrophysiological properties of hERG channel, but it does not seem to alter the cellular location of hERG channels. Thus, EGFP tagging to N-terminus might be used for research of subcellular location of hERG channels but not for the channel electrophysiological properties.     

Voltage-dependent calcium release in human malignant hyperthermia muscle fibers.

Malignant hyperthermia (MH) results from a defect of calcium release control in skeletal muscle that is often caused by point mutations in the ryanodine receptor gene (RYR1). In malignant hyperthermia-susceptible (MHS) muscle, calcium release responds more sensitively to drugs such as halothane and caffeine. In addition, experiments on the porcine homolog of malignant hyperthermia (mutation Arg615Cys in RYR1) indicated a higher sensitivity to membrane depolarization. Here, we investigated depolarization-dependent calcium release under voltage clamp conditions in human MHS muscle. Segments of muscle fibers dissected from biopsies of the vastus lateralis muscle of MHN (malignant hyperthermia negative) and MHS subjects were voltage-clamped in a double vaseline gap system. Free calcium was determined with the fluorescent indicator fura-2 and converted to an estimate of the rate of SR calcium release. Both MHN and MHS fibers showed an initial peak of the release rate, a subsequent decline, and rapid turn-off after repolarization. Neither the kinetics nor the voltage dependence of calcium release showed significant deviations from controls, but the average maximal peak rate of release was about threefold larger in MHS fibers.     

Two hydrophobic segments of the RTN1 family determine the ER localization and retention

Reticulon (RTN) proteins are localized to the endoplasmic reticulum (ER), and are related to intracellular membrane trafficking, apoptosis, inhibiting axonal regeneration, and Alzheimer's disease. The RTN proteins are produced without an N-terminal signal peptide. Their C-terminal domain contains two long hydrophobic segments. We analyzed the ER localization signal of human RTN1-A. Mutant proteins lacking the first (39 residues) or second (36 residues) hydrophobic segment showed ER localization. On the other hand, the mutant lacking both hydrophobic segments was cytosolic. Enhanced green fluorescent protein (EGFP) tagged with the first or second hydrophobic segment of RTN1-A was localized to the ER. These results suggest that each hydrophobic segment determines the ER localization. In addition, EGFP tagged with the truncated form of the first hydrophobic segment exhibited the localization to the Golgi rather than the ER. This suggests that the length of the hydrophobic segment contributes to the ER retention of RTN1-A.     

Multiple effects of caffeine on Ca2+ release and influx in human B lymphocytes

Caffeine has been used as a pharmacological tool to study the ryanodine receptor (RYR)-mediated Ca2+ release from caffeine-sensitive, inositol 1,4,5,-trisphosphate (IP3)-insensitive pools. In the present study, we demonstrate multiple effects of caffeine on Ca2+ homeostasis in human B lymphocytes. Although B cells express a functional RYR, which can be activated by 4-chloro-m-cresol following depletion of IP(3)-sensitive pools, caffeine does not activate RYR-mediated Ca2+ release. Instead, caffeine dose-dependently inhibited IP3 receptor (IP3R)-mediated Ca2+ release, RYR-mediated Ca2+ release and B cell receptor-initiated Ca2+ influx, while high concentrations of caffeine (> or = 25 mM) induced a Ca2+ influx. In contrast with its ability to suppress receptor-stimulated Ca2+ influx, caffeine had no significant effect on the store-operated Ca2+ (SOC) channel-dependent Ca2+ influx induced by thapsigargin. Thus, caffeine may act as an inhibitor on a single or multiple site(s) responsible for regulating the IP3R channel, RYR channel and presumably the receptor-mediated SOC channel. The present report may be the first demonstration of multiple effects of caffeine on Ca2+ mobilization in single cell type. Our results suggest the need for caution regarding use of caffeine simply as a RYR-activator to study Ca2+ homeostasis in eucaryotic cells.     

The Ile2453Thr mutation in the ryanodine receptor gene 1 is associated with facilitated calcium release from sarcoplasmic reticulum by 4-chloro-m-cresol in human myotubes

Central core disease (CCD) is a congenital disorder of skeletal muscle that is characterised histologically by typical central cores in type 1 skeletal muscle fibres. This disease is associated with malignant hyperthermia susceptibility and has been linked to the gene of skeletal muscle ryanodine receptor RYR1. In this study, we present a family with the spontaneous occurrence of the RYR1 Ile2453Thr mutation. Affected individuals were diagnosed as susceptible to malignant hyperthermia in the in vitro contracture test (IVCT) and showed histological signs of CCD. Myotubes were derived from the index patient. The calcium homeostasis in response to the ryanodine receptor agonist 4-chloro-m-cresol (4CmC) was investigated by calcium imaging using the Ca(2+)-sensitive fluorescent probe FURA 2. In the myotubes derived from the mutation carrier, the EC(50) of 4CmC was reduced to 94 micro as compared to 201 microM in a control group of 16 individuals non-susceptible to malignant hyperthermia. In the myotubes of the non-affected family members, the EC(50) was found within the same range as that of the control group. The reduction of EC(50) indicates a facilitated calcium release from sarcoplasmic reticulum in the myotubes of the index patient suggesting that the RYR1 Ile2453Thr mutation is pathogenic for the malignant hyperthermia susceptibility and CCD of the two affected individuals.     

Intracellular trafficking of a tagged and functional mammalian olfactory receptor.

Tagged G-protein-coupled receptors (GPCRs) have been used to facilitate intracellular visualization of these receptors. We have used a combination of adenoviral vector gene transfer and tagged olfactory receptors to help visualize mammalian olfactory receptor proteins in the normal olfactory epithelium of rats, and in cell culture. Three recombinant adenoviral vectors were generated carrying variously tagged versions of rat olfactory receptor I7. The constructs include an N-terminal Flag epitope tag (Flag:I7), enhanced green fluorescent protein (EGFP) fusion protein (EGFP:I7), and a C-terminal EGFP fusion (I7:EGFP). These receptor constructs were assayed in rat olfactory sensory neurons (OSNs) and in a heterologous system (HEK 293 cell line) for protein localization and functional expression. Functional expression of the tagged receptor proteins was tested by electroolfactogram (EOG) recordings in the infected rat olfactory epithelium, and by calcium imaging in single cells. Our results demonstrate that the I7:EGFP fusion protein and Flag:I7 are functionally expressed in OSNs while the EGFP:I7 fusion is not, probably due to inappropriate processing of the protein in the cells. These data suggest that a small epitope tag (Flag) at the N-terminus, or EGFP located at the C-terminus of the receptor, does not affect ligand binding or downstream signaling. In addition, both functional fusion proteins (Flag:I7 and I7:EGFP) are properly targeted to the plasma membrane of HEK 293 cells.     

Intracellular trafficking of a tagged and functional mammalian olfactory receptor

Tagged G‐protein‐coupled receptors (GPCRs) have been used to facilitate intracellular visualization of these receptors. We have used a combination of adenoviral vector gene transfer and tagged olfactory receptors to help visualize mammalian olfactory receptor proteins in the normal olfactory epithelium of rats, and in cell culture. Three recombinant adenoviral vectors were generated carrying variously tagged versions of rat olfactory receptor I7. The constructs include an N‐terminal Flag epitope tag (Flag:I7), enhanced green fluorescent protein (EGFP) fusion protein (EGFP:I7), and a C‐terminal EGFP fusion (I7:EGFP). These receptor constructs were assayed in rat olfactory sensory neurons (OSNs) and in a heterologous system (HEK 293 cell line) for protein localization and functional expression. Functional expression of the tagged receptor proteins was tested by electroolfactogram (EOG) recordings in the infected rat olfactory epithelium, and by calcium imaging in single cells. Our results demonstrate that the I7:EGFP fusion protein and Flag:I7 are functionally expressed in OSNs while the EGFP:I7 fusion is not, probably due to inappropriate processing of the protein in the cells. These data suggest that a small epitope tag (Flag) at the N‐terminus, or EGFP located at the C‐terminus of the receptor, does not affect ligand binding or downstream signaling. In addition, both functional fusion proteins (Flag:I7 and I7:EGFP) are properly targeted to the plasma membrane of HEK 293 cells. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 56–68, 2002     

Head-to-tail oligomerization of calsequestrin: a novel mechanism for heterogeneous distribution of endoplasmic reticulum luminal proteins

Many proteins retained within the endo/sarcoplasmic reticulum (ER/SR) lumen express the COOH-terminal tetrapeptide KDEL, by which they continuously recycle from the Golgi complex; however, others do not express the KDEL retrieval signal. Among the latter is calsequestrin (CSQ), the major Ca2+-binding protein condensed within both the terminal cisternae of striated muscle SR and the ER vacuolar domains of some neurons and smooth muscles. To reveal the mechanisms of condensation and establish whether it also accounts for ER/SR retention of CSQ, we generated a variety of constructs: chimeras with another similar protein, calreticulin (CRT); mutants truncated of COOH- or NH2-terminal domains; and other mutants deleted or point mutated at strategic sites. By transfection in L6 myoblasts and HeLa cells we show here that CSQ condensation in ER-derived vacuoles requires two amino acid sequences, one at the NH2 terminus, the other near the COOH terminus. Experiments with a green fluorescent protein GFP/CSQ chimera demonstrate that the CSQ-rich vacuoles are long-lived organelles, unaffected by Ca2+ depletion, whose almost complete lack of movement may depend on a direct interaction with the ER. CSQ retention within the ER can be dissociated from condensation, the first identified process by which ER luminal proteins assume a heterogeneous distribution. A model is proposed to explain this new process, that might also be valid for other luminal proteins.     

Caffeine Stimulates Amyloid β-Peptide Release from β-Amyloid Precursor Protein-Transfected HEK293 Cells

Abstract: Extracellular amyloid β-peptide (Aβ) deposition is a pathological feature of Alzheimer's disease and the aging brain. Intracellular Aβ accumulation is observed in the human muscle disease, inclusion body myositis. Aβ has been reported to be toxic to neurons through disruption of normal calcium homeostasis. The pathogenic role of Aβ in inclusion body myositis is not as clear. Elevation of intracellular calcium following application of calcium ionophore increases the generation of Aβ from its precursor protein (βAPP). A receptor-based mechanism for the increase in Aβ production has not been reported to our knowledge. Here, we use caffeine to stimulate ryanodine receptor (RYR)-regulated intracellular calcium release channels and show that internal calcium stores also participate in the genesis of Aβ. In cultured HEK293 cells transfected with βAPP cDNA, caffeine (5–10 m M ) significantly increased the release of Aβ fourfold compared with control. These actions of caffeine were saturable, modulated by ryanodine, and inhibited by the RYR antagonists ruthenium red and procaine. The calcium reuptake inhibitors thapsigargin and cyclopiazonic acid potentiated caffeine-stimulated Aβ release. NH₄Cl and monensin, agents that alter acidic gradients in intracellular vesicles, abolished both the caffeine and ionophore effects. Immunocytochemical studies showed some correspondence between the distribution patterns of RYR and cellular βAPP immunoreactivities. The relevance of these findings to Alzheimer's disease and inclusion body myositis is discussed.     

Simultaneous detection of DsRed2-tagged and EGFP-tagged human beta-interferons in the same single cells

The red fluorescent protein DsRed2 is a useful fusion tag for various proteins, together with the enhanced green fluorescent protein (EGFP). These chromoproteins have spectral properties that allow simultaneous distinctive detection of tagged proteins in the same single cells by dual color imaging. We used them for tagging a secretory protein, human interferon-beta (IFN-beta). Expression plasmids for human IFN-beta tagged with DsRed2 or with EGFP at the carboxyl terminal were constructed and their coexpression was examined in Mardin-Darby canine kidney epithelial cells. Although maturation of DsRed2 for coloration was slow and the color intensity was weak compared with EGFP, low temperature treatment (20 degrees C) allowed DsRed2-tagged human IFN-beta to be detected in the cells using color imaging. Consequently, the two chimeric proteins were shown to be colocalized in the same single cells by dual color confocal microscopy. This approach will be useful for investigating subcellular localization of not only cell resident proteins but also secretory proteins.     

Troponin T core structure and the regulatory NH2-terminal variable region

The conserved central and COOH-terminal regions of troponin T (TnT) interact with troponin C, troponin I, and tropomyosin to regulate striated muscle contraction. Phylogenic data show that the NH2-terminal region has evolved as an addition to the conserved core structure of TnT. This NH2-terminal region does not bind other thin filament proteins, and its sequence is hypervariable between fiber type and developmental isoforms. Previous studies have demonstrated that NH2-terminal modifications alter the COOH-terminal conformation of TnT and thin filament Ca2+-activation, yet the functional core structure of TnT and the mechanism of NH2-terminal modulation are not well understood. To define the TnT core structure and investigate the regulatory role of the NH2-terminal variable region, we investigated two classes of model TnT molecules: (1) NH2-terminal truncated cardiac TnT and (2) chimera proteins consisting of an acidic or basic skeletal muscle TnT NH2-terminus spliced to the cardiac TnT core. Deletion of the TnT hypervariable NH2-terminus preserved binding to troponin I and tropomyosin and sustained cardiac muscle contraction in the heart of transgenic mice. Further deletion of the conserved central region diminished binding to tropomyosin. The reintroduction of differently charged NH2-terminal domains in the chimeric molecules produced long-range conformational changes in the central and COOH-terminal regions to alter troponin I and tropomyosin binding. Similar NH2-terminal charge effects are demonstrated in naturally occurring cardiac TnT isoforms, indicating a physiological significance. These results suggest that the hypervariable NH2-terminal region modulates the conformation and function of the TnT core structure to fine-tune muscle contractility.     

Homogeneous Ca2+ stores in rat adrenal chromaffin cells

The localization and function of Ca(2+) stores in isolated chromaffin cells of rat adrenal medulla were investigated using confocal laser microscopy and amperometry. Binding sites for BODIPY-inositol 1,4,5-trisphosphate (IP(3)), -ryanodine (Ry), and -thapsigargin (Thap) were both perinuclear and at the cell periphery. The endoplasmic reticulum (ER), which was identified by ER Tracker dye, took up fluorescent Ry and IP(3), and the majority of BODIPY-Ry-binding area was bound by fluorescent IP(3). Under Ca(2+)-free conditions, the amount of caffeine-induced catecholamine secretion was 33% of that of muscarine-induced secretion, but muscarine induced little or no secretion after exposure to caffeine. Muscarine-induced Ca(2+) increases, as observed with fluo-3, lasted for a few tens of seconds under Ca(2+)-free conditions, whereas a caffeine-induced Ca(2+) transient diminished rapidly with a half decay time of 3s and this spike-like Ca(2+) transient was then followed by a sustained increase with a low level. These results indicate that IP(3) receptors and Ry receptors (RyRs) are present in common ER Ca(2+) storage and the lower potency of caffeine for secretion may be due to a rapid decrease in RyR channel activity to a low level.     

Caffeine-induced calcium release from the endoplasmic reticulum of acinar cells of the submandibular salivary gland

Ryanodine receptors (RyRs) play a key role in the generalization and spreading of calcium waves in excitable cells; however, the question of the existence of functionally active RyRs in nonexcitable cells demonstrating the capacity for exocytosis (e.g., salivary gland acini) remains open. We studied changes in the total amount of calcium stored in the endoplasmic reticulum (ER) of acinar cells of the submandibular salivary gland of rats and changes in the concentration of ionized Ca²⁺ inside the ER ([Ca²⁺]_ER) using, respectively, a metallochrome dye, arsenazo III, and a low-affinity fluorescent dye, mag-fura 2/AM. In permeabilized cells, caffeine caused dose-dependent decreases in the total amount of calcium and concentration of ionized calcium. The effective concentration of caffeine providing a 50% drop in the [Ca²⁺]_ER (EC₅₀) was, on average, 7.3 ± 1.1 mM. The caffeine-induced drop in the [Ca²⁺]^ER was insensitive to heparin; in addition, it was blocked by high concentrations (100 μM) of ryanodine, potentiated by ryanodine applied in mild concentrations (10 μM), and also demonstrated a bell-shaped dependence on the concentration of cytoplasmic Ca²⁺. Such peculiarities are typical characteristics of the RyR-mediated reaction. Therefore, functional RyRs whose activation results in a transient release of calcium from the ER are present in acinar cells of the submandibular salivary gland. Neirofiziologiya/Neurophysiology, Vol. 39, No. 2, pp. 107–112, March–April, 2007.     

Age-related alterations in caffeine-sensitive calcium stores and mitochondrial buffering in rat basal forebrain

The properties of caffeine- and thapsigargin-sensitive endoplasmic reticulum calcium stores were compared in acutely dissociated basal forebrain neurons from young and aged F344 rats by ratiometric microfluorimetry. The ability of these stores to sequester and release calcium resembles that observed in other central neurons, with an important role of mitochondrial calcium buffering in regulating the response to caffeine. An age-related reduction in the filling state of the stores in resting cells appears to be mediated by increased rapid calcium buffering, which reduces the availability of calcium for uptake into the stores. An age-related decrease in the amplitude of maximal caffeine-induced calcium release was attributed to increased mitochondrial buffering. There were no age-related differences in the sensitivity to caffeine or in the calcium sequestration/release process at the level of the endoplasmic reticulum per se. These findings demonstrate the importance of interactions between cellular calcium buffering mechanisms and provide details regarding age-related changes in calcium homeostasis which have been thought to occur in these and other neurons associated with age-related neuronal dysfunctions.     

Involvement of calcium mobilization from caffeine-sensitive stores in mechanically induced cell cycle arrest in the dinoflagellate Crypthecodinium cohnii

Mechanical loads can profoundly alter cell growth and cell proliferation. The dinoflagellates are especially sensitive to mechanical stimulation. Many species will be arrested in cell cycle in response to turbulence or shear stress. We demonstrate here that mechanical shaking and caffeine, the ryanodine-receptor agonist, induced an elevation of cytosolic calcium in the dinoflagellate Crypthecodinium cohnii. Dantrolene, a ryanodine-receptor antagonist, dose-dependently inhibited both shaking-induced and caffeine-induced calcium release. Similar to the effect of mechanical shaking, caffeine alone dose-dependently and reversibly induced cell cycle arrest in dinoflagellates. Prolonged shaking substantially abolished the magnitude of caffeine-induced calcium release and vice-versa, suggesting that both agents released calcium from similar stores through ryanodine receptors. Fluorescence-conjugated ryanodine gave positive labeling, which could be blocked by ryanodine, in the cortice of C. cohnii cells. In addition, caffeine or shaking mobilized intracellular chlortetracycline (CTC)-positive membrane-bound calcium, which could be similarly depleted by t-BuBHQ, a SERCA pump inhibitor. Prior treatment with shaking or caffeine also inhibited the ability of the other agent in mobilizing CTC-positive calcium. CTC-positive microsomal fractions could also be induced to release calcium by caffeine and cADPR, the ryanodinee receptor modulator. t-BuBHQ, but not calcium ionophores, induced cell cycle arrest, and the calcium chelator BAPTA-AM was unable to rescue caffeine-induced cell cycle arrest. These data culminate to suggest that mobilization or depletion of caffeine-sensitive calcium stores, but not calcium elevation per se, is involved in the induction of cell cycle arrest by mechanical stimulation. The present study establishes the role of caffeine-sensitive calcium stores in the regulation of cell cycle progression.     

The effect of verdazyl radicals on Ca2+ metabolism in preparations of heavy sarcoplasmic reticulum

Three verdazyl radicals were studied for their effect on calcium accumulation and outflux (passive and Ca- or caffeine-induced) and conformational state of the Ca-ATPase. All three compounds differently affected the ATP-dependent Ca-accumulation. Their effect on the Ca-release from the sarcoplasmic reticulum vesicles could not be explained by their influence on the Ca-accumulation system. The Ca2+ amount liberated by the calcium or caffeine addition was equal in both cases but was modified differently by the used verdazyl compounds. The data obtained suggest that Ca-induced and caffeine-induced calcium release is realized by different mechanisms.     

Expression of the ryanodine receptor isoforms in immune cells.

Ryanodine receptor (RYR) is a Ca(2+) channel that mediates Ca(2+) 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 1 RYR (RYR1) was expressed in CD19(+) B lymphocytes, but less frequently in CD3(+) T lymphocytes and in CD14(+) monocytes. 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 protein-1alpha (MIP1alpha) or TGF-beta. 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 Ca(2+) release and thereby confirmed functional expression of the RYR in the cell lines expressing RYR mRNA. Moreover, concordant induction of RYR mRNA with Ca(2+) channel function was found in Jurkat T cells. In untreated Jurkat T cells, 4CmC (>1 mM) had no effect on Ca(2+) release, whereas 4CmC (<400 microM) caused Ca(2+) release after the induction of RYR2 and RYR3 that occurred after treatment with stromal cell-derived factor 1, macrophage-inflammatory protein-1alpha, or TGF-beta. Our results demonstrate expression of all three isoforms of RYR mRNA in hemopoietic cells. Induction of RYRs in response to chemokines and TGF-beta suggests roles in regulating Ca(2+)-mediated cellular responses during the immune response.