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* Inflammation Research, Pharmacology Laboratories,
Molecular Medicine Research, Molecular Medicine Laboratories, and
Medicinal Chemistry Research II, Chemistry Laboratories, Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd., Tsukuba-shi, Ibaraki, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1. It was suggested that YM-58483 inhibited Ca2+ influx through SOC channels without affecting the TCR signal transduction cascade. Furthermore, YM-58483 inhibited thapsigargin-induced sustained Ca2+ influx with an IC50 value of 100 nM without affecting membrane potential. YM-58483 inhibited by 30-fold the Ca2+ influx through SOC channels compared with voltage-operated Ca2+ channels, while econazole inhibited both SOC channels and voltage-operated Ca2+ channels with an equivalent range of IC50 values. YM-58483 potently inhibited IL-2 production and NF-AT-driven promoter activity, but not AP-1-driven promoter activity in Jurkat cells. Moreover, this compound inhibited delayed-type hypersensitivity in mice with an ED50 of 1.1 mg/kg. Therefore, we concluded that YM-58483 was a novel store-operated Ca2+ entry blocker and a potent immunomodulator, and could be useful for the treatment of autoimmune diseases and chronic inflammation. Furthermore, YM-58483 would be a candidate for the study of capacitative Ca2+ entry mechanisms through SOC/CRAC channels and for identification of putative Ca2+ channel genes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we investigated the effects of YM-58483, a pyrazole derivative, on Ca2+ influx and IL-2 production in Jurkat cells in vitro, and on T lymphocyte-mediated immune responses in vivo using a mouse model of delayed-type hypersensitivity (12).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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YM-58483, 4-methyl-4'-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]-1,2,3-thiadiazole-5-carboxanilide, and SK&F-96365 were synthesized by Yamanouchi Pharmaceutical (Tokyo, Japan). YM-58483 was identified from the screen of a chemical library in a high throughput screening to find novel agents of inhibiting thapsigargin-induced sustained Ca2+ influx in Jurkat cells. Cyclosporin A was purchased from Sandoz (Basel, Switzerland). Econazole, ionomycin, nifedipine, thapsigargin, PMA, and penicillin-streptomycin solution were purchased from Sigma-Aldrich (St. Louis, MO). Fura 2-AM was purchased from Dojindo Laboratories (Kumamoto, Japan). Prednisolone, 2,4,6-trinitrochlorobenzene (TNCB), EGTA, NaCl, KCl, MgCl2 6H2O, CaCl2 2H2O, D(+)-glucose, and HEPES were obtained from Nacalai Tesuque (Kyoto, Japan). DMSO, acetone, and ethanol were purchased from Kanto Chemical (Tokyo, Japan). Methylcellulose (MC) was obtained from Shin-Etsu Chemical (Tokyo, Japan). Human anti-CD3 mAb, UCHT1, and C105 were purchased from R&D Systems (Minneapolis, MN) and Leinco Technologies (St. Louis, MO), respectively. Anti-phospholipase C1 (PLC1) mAb and anti-tyrosine phosphorylation mAb (4G10) conjugated with HRP were bought from Upstate Biology (Charlottesville, VA). Protease inhibitor mixture was obtained from Wako Pure Chemical Industries (Tokyo, Japan). Sheep anti-mouse Ig Ab conjugated with HRP and protein G-Sepharose gel (4 Fast Flow) were obtained from Amersham Pharmacia Biotech AB (Uppsala, Sweden). Anti-NF-ATc2 (4G6-G5.1) mAb was purchased from BD PharMingen (San Diego, CA). RPMI 1640 medium, DMEM/F-12 medium, and heat-inactivated horse serum were purchased from Life Technologies (Grand Island, NY). FBS was obtained from JRH Biosciences (Lenexa, KS). Each chemical compound was dissolved in DMSO for experiments in vitro. TNCB was dissolved in 7% (w/v) and 0.25% (w/v) with a solvent containing 10% acetone and 90% ethanol. YM-58483 and prednisolone were suspended in 0.5% MC for oral administration.
Econazole (13) and SK&F-96365 (14) were used as reference compounds for the blocker of SOC channels, and nifedipine was used as a reference compound for the blocker of voltage-operated Ca2+ (VOC) channels (15).
Cell culture
Human T cell leukemia, Jurkat cells (E6-1) were purchased from American Type Culture Collection (Manassas, VA), and were cultured in RPMI 1640 medium with 10% FBS and a 100 U/ml penicillin-streptomycin solution in a 37°C, humidified 5% CO2 incubator (CPD-170W; Hirasawa Works, Tokyo, Japan). A stable IL-2 reporter cell line, IL-2/Jurkat cells (16), and a murine neuroendocrine cell line, PC12-h cells (17), were kindly donated by Kuromitsu and S. Hashimoto, respectively (Yamanouchi Pharmaceutical). PC12-h cells were cultured in rat type I collagen-coated flasks (Biocoat; BD Biosciences, Bedford, MA) in DMEM/F-12 medium with 10% horse serum, 7% FBS, and 100 U/ml penicillin and streptomycin in a 37°C, humidified 5% CO2 incubator.
Measurement of intracellular Ca2+ by spectrofluorometer
Jurkat cells (2 x 106 cells/ml) were suspended in HBSS of the following composition (mM): NaCl, 137; KCl, 5.8; MgCl2, 1; CaCl2, 2.5; glucose, 5; and HEPES, 10, pH 7.4, and were loaded with 1 µM fura 2-AM at room temperature for 45 min. Following centrifugation (200 x g, 24°C for 3 min, himac CF 7D2; Hitachi, Tokyo, Japan), cells were successively washed to remove unincorporated dye and resuspended in Ca2+-free HBSS. The fluorescence of the cell suspension was monitored by spectrofluorometer (CAF-110; JASCO, Tokyo, Japan) at an emission wavelength of 500 nm and excitation wavelengths of 340 and 380 nm, respectively. The concentration of intracellular Ca2+ was calculated from the 340/380 nm fluorescence ratio (R) with the standard equation (18). The Rmax value was obtained from the 25 µM ionomycin-induced fluorescence ratio, and the Rmin value from the fluorescence ratio on additional treatment with 50 mM EGTA after ionomycin. A dissociation constant (Kd) of 224 nM was used for the calculation of intracellular Ca2+.
Anti-CD3 mAb-induced increase in intracellular Ca2+
TCR-cross talk-linked intracellular Ca2+ responses were induced by 10 µg/ml human anti-CD3 mAb (UCHT1). Each concentration of compound was added 1 min before TCR stimulation in the absence or presence of extracellular Ca2+ (2 mM CaCl2).
Thapsigargin-induced increase in intracellular Ca2+
To investigate the inhibitory effects of compounds on Ca2+ influx through SOC channels, Ca2+ influx was evoked by 1 µM thapsigargin in Jurkat cells. 1) For the pretreatment experiment, Jurkat cells were stimulated by thapsigargin in Ca2+-free HBSS. Each concentration of compound was added 10 min after thapsigargin, and Ca2+ influx was evoked by addition of exogenous 2 mM CaCl2. 2) For the posttreatment study, each concentration of compound was added after a sustained intracellular Ca2+ influx had been evoked in the presence of extracellular Ca2+ 10 min after thapsigargin stimulation.
Measurement of intracellular Ca2+ in 96-well microplates
A 96-well microplate assay system was used in the comparative study on channel inhibition. Fluorescence of fura 2 was measured in a fluorescence microplate reader (Fluostar; SLT Labinstruments, Salzburg, Austria) with an excitation wavelength of 340 and 380 nm at an emission wavelength of 500 nm. The concentration of intracellular Ca2+ in each well was calculated from the fluorescence ratio with the standard equation. The Rmax value was obtained from 25 µM ionomycin-treated wells. The Rmin value was obtained from 25 µM ionomycin- and 50 mM EGTA-treated wells.
Comparative study on the inhibition of SOC channels
Inhibition of SOC channels was evaluated in Jurkat cells (2 x 106 cells/ml). In 96-well microplates (MS-8496K; Sumitomo Bakelite, Tokyo, Japan), 200 µl of fura 2-loaded Jurkat cells was stimulated with 1 µM thapsigargin for 30 min, and the intracellular Ca2+ concentration was measured at an endpoint of 30 min. Each compound was added at the same time during thapsigargin stimulation.
Comparative study on the inhibition of VOC channels
It was noted that L-type VOC channels were expressed in PC12 cells (19). The inhibition of VOC channels was assessed in PC12-h cells (1 x 106 cells/ml) after detachment from the flask using the protein digestive enzyme, Actinase E (Kaken Pharmaceutical, Tokyo, Japan), and fura 2 loading. In 96-well microplates, 200 µl of fura 2-loaded PC12-h cells was stimulated with 50 mM KCl for 20 min, and the intracellular Ca2+ concentration was measured at the endpoint of 20 min.
Measurement of membrane potential
Membrane potential was measured using the fluorometric imaging plate reader (FLIPR; Molecular Devices, Sunnyvale, CA). Jurkat cells (1 x 107/ml) were suspended in HBSS buffer, and mixed with an equal amount of the membrane potential dye (FLIPR membrane potential assay kit; Molecular Devices). Then 90 µl of cells and 90 µl of dye were added to each well of 96-well black/clear-bottom microplate (BD Biosciences), and incubated at 37°C for 30 min before experiment. Laser intensity of the FLIPR was set to provide a basal fluorescence signal of 2000 relative fluorescence, and measurements of fluorescence were taken at 6-s interval for the first 3 min, then at 20-s intervals for the remaining 8 min. An on-board 96-well pipettor allowed simultaneous addition of compound concentrations (20 µl) delivered at a rate of 10 µl/s, 10 s after the start of readings. Fluorescence changes were captured by a cooled charge-coupled device camera and integrated to an on-line personal computer. The depolarization level was presented as percentage of 50 mM KCl-induced response using values of 10 min after induction.
Immunoprecipitation assay and Western blotting
Jurkat cells (4 x 107 cells/ml) were treated with varying concentration of compounds for 10 min at room temperature. The cells were stimulated with 3 µg/ml anti-CD3 mAb (C105) for 2 min, and solubilized in Triton X-100 lysis buffer (0.5% Triton X-100, 1 mM Na3VO4, 150 mM NaCl, 50 mM NaF, 10 mM EDTA, and 50 mM Tris, pH 7.5) containing protease inhibitors. The cell lysate was centrifuged at 15,000 x g for 20 min. Clarified lysate was incubated for 1 h at 4°C with 1 µg of anti-PLC1 mAb and 40 µl of 50% slurry of protein G-Sepharose. After washing, immunoprecipitates were subjected to SDS-PAGE and transferred to nitrocellulose membrane (Amersham Pharmacia Biotech AB). Membranes were preincubated with BlockAce (Dainippon Pharmaceutical, Tokyo, Japan), followed by immunoblotting with HRP-conjugated anti-phosphotyrosine mAb for 1 h at room temperature. Subsequently, the membranes were reblotted with anti-PLC1 mAb and HRP-conjugated anti-mouse Ig Ab. Membranes were developed using ECL (Amersham Pharmacia Biotech AB). Data were analyzed using ImageQuaNT software (Molecular Dynamics, Sunnyvale, CA).
IL-2 production assay
Jurkat cells (5 x 106 cells/ml) were placed in a 96-well microplate and incubated with 20 µg/ml PHA (Sigma-Aldrich) in the CO2 incubator for 20 h, and the supernatant was collected from these cells after centrifugation (200 x g, 24°C for 3 min). The concentration of IL-2 in each supernatant was measured by the human IL-2 ELISA system (human IL-2 ELISA Kit DuoSet; Genzyme, Cambridge, MA). OD values at 450 nm were measured by microplate reader (Spectra Max 190; Molecular Devices).
IL-2 reporter gene expression assay
IL-2 gene expression was evaluated in a stable IL-2 reporter cell line, IL-2/Jurkat cells (16), established from Jurkat cells with a reporter plasmid pGVIL-2 and the promoter region of the IL-2 gene. IL-2/Jurkat cells (2 x 106 cells/ml) were placed in a 96-well microplate (Nunclon 
Surface; Nagle Nunc International, Rochester, NY), and were stimulated with 20 µg/ml PHA in the CO2 incubator for 20 h. YM-58483 and other compounds were added at the same time during PHA stimulation at 0.25% DMSO. In this assay, 0.25% DMSO did not influence PHA-stimulated IL-2 reporter activity in IL-2/Jurkat cells. The reaction was stopped by addition of 50 µl of solubilization buffer (10 mM Tris-HCl/pH 7.8, 0.5 mM MgCl2, 10 mM DTT, and 0.1% Triton X-100). Luciferase activities were measured with a ML3000 luminometer (Dynatech Laboratories, Chanitilly, VA) after addition of 50 µl of substrate solution (5 mM luciferin, 2 mM coenzyme A, 2 mM ATP, 0.5 mM MgCl2, and 2 mM Mg(OH)2 in 10 mM Tris-HCl/pH 7.8 solution). Luciferase activities were normalized by the ratio with a nonstimulated activity, and were represented by the fold induction.
NF-AT and AP-1 reporter gene assays
Jurkat cells (1 x 107 cells/0.4 ml) were transfected with 40 µg of pGL-TATA, pGL(AP-1)4, or pGL(NF-AT)3 by means of electroporation under the condition of 280 V and 960 µF in 0.4-cm cuvettes (Gene Pulser Cuvette; Bio-Rad Laboratories, Hercules, CA). After 24-h transfection, pGL-TATA-transfected cells were stimulated with 10 ng/ml PMA and 1 µM ionomycin, or 10 ng/ml PMA, pGL(AP-1)4-transfected cells were stimulated with PMA, and pGL(NF-AT)3-transfected cells were stimulated with PMA and ionomycin for 16 h. Compounds were added at the same time during stimulation at 0.1% DMSO. Luciferase activities were measured the same as IL-2 reporter gene assay. Each reporter plasmid was kindly provided by S. Kuromitsu (16).
NF-AT dephosphorylation assay
Jurkat cells (1 x 107 cells/ml) were tested with varying concentration of compounds for 30 min at 37°C. The cells were stimulated with 1 µM ionomycin for 30 min at 37°C. After stimulation, the cells were centrifuged at 200 x g for 2 min, and were solubilized in 100 µl of Triton X-100 lysis buffer. The cell lysate was centrifuged at 15,000 x g for 20 min; the clarified lysate was subjected to SDS-PAGE; and NF-ATc2 was detected by Western blotting with anti-NF-ATc2 mAb.
TNCB-induced contact hypersensitivity in mice
Five-week-old male CD-1 mice were obtained from Japan SLC (Shizuoka, Japan). The animals were treated with 100 µl of a 7% TNCB solution in the abdominal region after cutting their abdominal hair under anesthesia at day 0. Seven days after the TNCB sensitization, the thickness of both ears was measured, and 10 µl of 0.25% TNCB was applied both inside and outside of the ear pinnas. Only a solvent was applied for negative control mice. Ear thickness was measured using a dial thickness gauge (Peacock; Ozaki MFG, Tokyo, Japan) 24 h after the TNCB challenge, and changes in swelling were calculated from the value pre-exposure. YM-58483 (1–30 mg/kg) and prednisolone (0.3–3 mg/kg) were administered orally 1 h before exposure to TNCB, and 0.5% MC was administered orally in negative control and control animals.
Statistical analysis
Data are represented by the mean ± SE. Statistical analyses were performed by SAS (Cary, NC). Percent inhibition of Ca2+ influx, IL-2 production, and IL-2 gene expression was calculated for each concentration of compound as follows: ((solvent control - compound group)/(solvent control - nonstimulation control)) x 100. The IC50 was calculated from the percent inhibition value for nonstimulation and control stimulation, and the ED50 was calculated from the value for the negative control and control. Statistical analysis of the effect was performed by Student’s t test or Dunnett’s multiple range test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Anti-CD3 mAb (10 µg/ml) induced a transient and then sustained increase in intracellular Ca2+ in the presence of extracellular Ca2+. YM-58483 (3 µM) and econazole (3 µM) partially inhibited the transient increase, and completely inhibited the sustained increase (Fig. 1A). In contrast, anti-CD3 mAb evoked only a transient increase in intracellular Ca2+ in the absence of extracellular Ca2+, but the maximum Ca2+ concentration was evidently diminished by the compound-induced inhibition in the presence of extracellular Ca2+ (Fig. 1, A and B). Neither YM-58483 nor econazole inhibited the transient increase in intracellular Ca2+ in the absence of extracellular Ca2+ (Fig. 1B). Moreover, YM-58483 (3 µM) did not affect the baseline intracellular Ca2+ in Jurkat cells (Fig. 2).
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YM-58483 (0.3–3 µM) and econazole (0.3–3 µM) did not affect the anti-CD3 mAb-induced tyrosine phosphorylation of PLC1 in Jurkat cells (Fig. 3, A and C). The amount of PLC1 protein did not change on stimulation with anti-CD3 or any compound (Fig. 3B).
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Thapsigargin evoked a transient increase in intracellular Ca2+ in the absence of extracellular Ca2+, and the subsequent addition of 2 mM CaCl2 induced a sustained Ca2+ influx (Fig. 4). Pretreatment with YM-58483 (0.01–10 µM) and econazole (0.1–10 µM) 1 min before addition of 2 mM CaCl2 inhibited the sustained Ca2+ influx in a concentration-dependent manner with IC50 values of 100 and 680 nM, respectively (Figs. 4 and 5; Table I). Furthermore, YM-58483 and econazole reduced concentration dependently the already evoked Ca2+ influx with the equivalent IC50 values compared with pretreatment (Figs. 5 and 6; Table I).
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YM-58483 inhibited thapsigargin-induced Ca2+ influx through SOC channels in Jurkat cells with an IC50 value of 150 ± 11 nM, which was equivalent to the value for the spectrofluorometer system (Fig. 7A; Table II). The VOC channel blocker, nifedipine, potently inhibited the high concentration KCl-induced Ca2+ influx through VOC channels in PC12-h cells with an IC50 of 2.7 ± 0.41 nM, but not that through SOC channels (2.0% at 10 µM). The IC50 value of YM-58483 for VOC channels was 4700 ± 550 nM, which was 30-fold less than that for SOC channels (Fig. 7B; Table II). Econazole and SK&F-96365 showed no selectivity for SOC channels and VOC channels (Table II).
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The high concentration KCl (50 mM) caused remarkable depolarization in Jurkat cells (Fig. 8). YM-58483 (3 and 10 µM) did not affect membrane potential (Fig. 8, A and C). In contrast, econazole (3 and 10 µM) caused slight, but significant depolarization in a concentration-dependent manner (Fig. 8, B and C).
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The supernatant from Jurkat cells stimulated with PHA for 20 h contained a significant amount of IL-2 compared with that from nonstimulated cells (PHA (-), 15 pg/ml; PHA (+), 2126 pg/ml; p < 0.01 by Student’s t test, n = 10). YM-58483 and cyclosporin A inhibited this IL-2 production in a concentration-dependent manner with IC50 values of 17 ± 3.2 and 3.2 ± 0.45 nM, respectively (Fig. 9A; Table III). Econazole and SK&F-96365 also inhibited IL-2 production in a concentration-dependent manner, but these inhibitory effects were weaker than that of YM-58483 (Fig. 9B; Table III). The VOC channel blocker, nifedipine, did not inhibit IL-2 production from Jurkat cells.
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YM-58483, econazole, and cyclosporin A inhibited IL-2 reporter gene expression in a concentration-dependent manner with IC50 values of 10 ± 3.1, 1700 ± 350, and 4.8 ± 1.8 nM, respectively (Fig. 10; Table IV). SK&F-96365 also inhibited IL-2 reporter gene expression in a concentration-dependent manner (51% at 10 µM). YM-58483 and cyclosporin A inhibited PMA/ionomycin-induced NF-AT reporter gene activity in a concentration-dependent manner in pGL(NF-AT)3-transfected Jurkat cells (Fig. 11). In contrast, these compounds did not affect PMA-induced AP-1 reporter gene activity in pGL(AP-1)4-transfected cells. Then pGL-TATA reporter gene activity was not affected by the stimulation of PMA/ionomycin or PMA.
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YM-58483 (0.03–3 µM) and cyclosporin A (0.01–1 µM) inhibited PMA/ionomycin-induced dephosphorylation of NF-AT in a concentration-dependent manner in Jurkat cells (Fig. 12).
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TNCB-induced ear swelling was significantly increased in control animals compared with the vehicle-challenged negative control (Fig. 13). YM-58483 and prednisolone inhibited dose dependently TNCB-induced ear swelling in sensitized mice with ED50 values of 1.1 and 3.7 mg/kg, respectively (Fig. 13).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 (20), which leads to the formation of IP3 and is followed by the stimulation of IP3-sensitive Ca2+ stores and an increase in intracellular Ca2+. In the presence of extracellular Ca2+, anti-CD3 mAb evoked a transient peak, followed by a sustained increase in intracellular Ca2+. In contrast, anti-CD3 mAb evoked only a transient increase in intracellular Ca2+ in the absence of extracellular Ca2+, in which the maximum Ca2+ concentration was evidently diminished by the levels of econazole-induced inhibition observed in the presence of extracellular Ca2+. It is noted that econazole is a blocker of store-operated Ca2+ entry through SOC channels (21), and inhibits ICRAC interacting with the outside of the membrane in lymphocytes (22). These results suggest that the transient peak of intracellular Ca2+ observed in the presence of extracellular Ca2+ consists of two components, one derived from Ca2+ stores and one from the extracellular environment. In the presence of extracellular Ca2+, YM-58483 and econazole partially, but significantly inhibited the anti-CD3 mAb-induced transient increase in intracellular Ca2+, and YM-58483 completely inhibited the sustained Ca2+ influx. YM-58483 and econazole did not inhibit the transient increase in intracellular Ca2+ in the absence of extracellular Ca2+. Moreover, these compounds did not affect the phosphorylation of PLC1 on TCR stimulation. Therefore, it is suggested that YM-58483 as well as econazole inhibit the sustained Ca2+ influx without affecting the TCR signal transduction cascade leading to the stimulation of IP3-sensitive Ca2+ stores. YM-58483 and econazole inhibited a sustained Ca2+ influx following pretreatment, and reduced the already evoked sustained Ca2+ influx after posttreatment. When YM-58483 interacts with some unidentified cytosolic molecules, which regulate Ca2+ influx from SOC channels, the inhibitory effects of these compounds on posttreatment must be weaker than that pretreatment and must indicate a slow reducing pattern. But, in the present study, YM-58483 and econazole inhibited a sustained Ca2+ influx both pre- and posttreatment to the same degree. Moreover, YM-58483 and econazole rapidly reduced the sustained Ca2+ influx posttreatment. It is noted that membrane depolarization reduces Ca2+ influx in lymphocytes (7). Indeed, it was reported that KCl-induced membrane depolarization inhibited Ca2+ influx in the CD3-activated Jurkat cells (18). In the present study, YM-58483 did not change membrane potential in Jurkat cells. In contrast, econazole caused slight, but significant depolarization in a concentration-dependent manner. These results indicated that YM-58483 inhibited Ca2+ influx without affecting membrane potential, and econazole inhibited Ca2+ influx partly through membrane depolarization. Therefore, it is suggested that YM-58483 inhibited store-operated Ca2+ entry by directly interacting with SOC channels or channel-regulatory cofactors at the membrane surface without affecting the depletion of Ca2+ stores.
It has been reported that econazole and SK&F-96365 inhibited store-operated Ca2+ influx through SOC channels (13, 14, 22, 23). Indeed, econazole and SK&F-96365 inhibited Ca2+ influx through SOC channels in lymphocytes, but these compounds also inhibited Ca2+ influx through VOC channels in PC12-h cells over the same range of concentrations. In contrast, the effect of YM-58483 on SOC channels was 30 times more selective against Ca2+ influx than that through VOC channels, indicating that YM-58483 is a selective SOC channel blocker. These results suggest that YM-58483 is a novel Ca2+ entry blocker, which selectively inhibits Ca2+ through SOC channels, not VOC channels.
Ca2+ influx through SOC channels is a key event for immune responses, including IL-2 production in lymphocytes. The IL-2 gene is up-regulated by NF-AT, which needs a sustained increase of intracellular Ca2+ via the activation of SOC channels (4). YM-58483 inhibited IL-2 production from PHA-stimulated Jurkat cells as potently as cyclosporin A. It is noted that cyclosporin A inhibits the interaction of calcineurin with NF-AT, and calcineurin is activated by Ca2+ and calmodulin (5, 6). In our preliminary studies, cyclosporin A, unlike YM-58483, did not inhibit the store-operated Ca2+ influx in Jurkat cells (data not shown). Furthermore, YM-58483 as well as cyclosporin A inhibited IL-2 reporter gene expression in PHA-stimulated stable IL-2/Jurkat cells. Econazole and SK&F-96365 also inhibited IL-2 production and IL-2 reporter gene expression. Therefore, it is suggested that YM-58483 inhibits the transcription of NF-AT and production of IL-2 by inhibiting Ca2+ influx. Indeed, YM-58483 inhibited NF-AT dephosphorylation and NF-AT reporter gene activity without affecting AP-1 reporter gene activity as well as cyclosporin A in the present study. It is noted that AP-1 activation is regulated by Ca2+-independent Ras/Raf pathway (24, 25), which supports that YM-58483 did not affect PMA-induced AP-1 reporter gene activity in the present study. TCR-mediated signaling is coupled to the downstream phosphorylation of PLC1, which activates both PI3 and Ras/Raf pathways (26, 27). YM-58483 did not inhibit PMA/ionomycin-induced phosphorylation of PLC1 and TCR-stimulated transient Ca2+ efflux from Ca2+ store, indicating that YM-58483 regulates the activation of NF-AT without affecting TCR signaling. Therefore, it is suggested that the inhibition of sustained Ca2+ influx is main mechanism of the immune modulatory action of this compound. Furthermore, YM-58483 as well as prednisolone orally inhibited TNCB-induced contact hypersensitivity in mice. The contact hypersensitivity is regarded as a prototype of T lymphocyte-mediated delayed-type hypersensitivity reactions (12). This suggested that YM-58483 could be a useful immune modulator in autoimmune diseases or chronic inflammation. Therefore, it is concluded that YM-58483 is an in vivo effective novel Ca2+ entry blocker, and is a new-type immune modulatory agent via the different mechanism from well-known immunosuppressants such as cyclosporin A or FK506.
It was recently reported that PHA stimulates the increased number of SOC channels in T lymphocytes (11), indicating that intracellular Ca2+ signaling is enhanced in activated T lymphocytes, and inhibition of Ca2+ influx is important for the regulation of T lymphocytes. In the present study, YM-58483 did not affect baseline intracellular Ca2+ levels, as shown in Fig. 2, suggesting that YM-58483 would not influence resting lymphocytes. Therefore, YM-58483 could be a potent regulator for the activation of T lymphocytes, and could be useful for the treatment of autoimmune diseases and chronic inflammation. Furthermore, YM-58483 could be a useful tool for the study of capacitative Ca2+ entry mechanisms through SOC/CRAC channels and putative Ca2+ entry channel genes.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: IP3, inositol triphosphate; CRAC, Ca2+ release-activated Ca2+; FLIPR, fluorometric imaging plate reader; MC, methylcellulose; PLC1, phospholipase C1; SOC, store-operated Ca2+; TNCB, 2,4,6-trinitrochlorobenzene; VOC, voltage-operated Ca2+.
Received for publication April 10, 2002. Accepted for publication February 18, 2003.
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References |
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1 (PLC-1) to T-cell antigen receptor-mediated signaling: reconstitution studies of a PLC-1-deficient Jurkat T-cell line. Mol. Cell. Biol. 20:9149.This article has been cited by other articles:
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  S. B. Semenova, I. O. Vassilieva, A. F. Fomina, A. L. Runov, and Y. A. Negulyaev Endogenous expression of TRPV5 and TRPV6 calcium channels in human leukemia K562 cells Am J Physiol Cell Physiol, May 1, 2009; 296(5): C1098 - C1104. [Abstract] [Full Text] [PDF]
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 S. Kiyonaka, K. Kato, M. Nishida, K. Mio, T. Numaga, Y. Sawaguchi, T. Yoshida, M. Wakamori, E. Mori, T. Numata, et al. Selective and direct inhibition of TRPC3 channels underlies biological activities of a pyrazole compound PNAS, March 31, 2009; 106(13): 5400 - 5405. [Abstract] [Full Text] [PDF]
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 B. A. Kachoei, R. J. Knox, D. Uthuza, S. Levy, L. K. Kaczmarek, and N. S. Magoski A Store-Operated Ca2+ Influx Pathway in the Bag Cell Neurons of Aplysia J Neurophysiol, November 1, 2006; 96(5): 2688 - 2698. [Abstract] [Full Text] [PDF]
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J. R. H. Mauban, K. Wilkinson, C. Schach, and J. X.-J. Yuan Histamine-mediated increases in cytosolic [Ca2+] involve different mechanisms in human pulmonary artery smooth muscle and endothelial cells Am J Physiol Cell Physiol, February 1, 2006; 290(2): C325 - C336. [Abstract] [Full Text] [PDF]
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C. Zitt, B. Strauss, E. C. Schwarz, N. Spaeth, G. Rast, A. Hatzelmann, and M. Hoth Potent Inhibition of Ca2+ Release-activated Ca2+ Channels and T-lymphocyte Activation by the Pyrazole Derivative BTP2 J. Biol. Chem., March 26, 2004; 279(13): 12427 - 12437. [Abstract] [Full Text] [PDF]
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, 0.01–10 µM) and econazole (•, 0.03–10 µM) on exogenous 2 mM Ca2+ induced sustained Ca2+ influx in thapsigargin-stimulated cells (see Fig. 3
, 0.003–10 µM), econazole (
, 0.003–10 µM), SK&F-96365 (
, 0.003–10 µM) on Ca2+ influx through SOC channels induced by 1 µM thapsigargin in Jurkat cells. B, Effects of YM-58483 (
; 10 µM, 
; 10 µM, 
