Identification and Functional Characterization of Voltage-dependent Calcium Channels in T Lymphocytes
2003; Elsevier BV; Volume: 278; Issue: 47 Linguagem: Inglês
10.1074/jbc.m309268200
ISSN1083-351X
AutoresMaya F. Kotturi, Douglas A. Carlow, Junella C. Lee, Hermann J. Ziltener, Wilfred A. Jefferies,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoIn T lymphocytes, sustained calcium (Ca2+) influx through Ca2+ channels localized in the plasma membrane is critical for T cell activation and proliferation. Previous studies indicated that voltage-dependent Ca2+ channels (VDCCs) play a role in Ca2+ mobilization during T lymphocyte activation. However, the role of VDCCs in otherwise nonexcitable cells is still poorly understood. We used RT-PCR to identify a transcript encoding the pore-forming α1F-subunit of an L-type Ca2+ channel in T lymphocytes. Its identity was confirmed by DNA sequencing. To further investigate the contribution of Ca2+ influx through VDCCs, we assessed the effects of the 1,4-dihydropyridine L-type Ca2+ channel agonist, (+/-) Bay K 8644, and antagonist, nifedipine, on the human Jurkat T cell leukemia line, human peripheral blood T lymphocytes and mouse splenocytes. We found that treatment of T lymphocytes with (+/-) Bay K 8644 increased intracellular Ca2+ and induced the activation of phosphoextracellular-regulated kinase 1/2 (Erk1/2), whereas nifedipine blocked Ca2+ influx, the activity of Erk1/2 and nuclear factor of activated T cells (NFAT), interleukin-2 (IL-2) production, and IL-2 receptor expression. Nifedipine also significantly suppressed splenocyte proliferation in an in vitro mixed lymphocyte reaction and the proliferation of male antigen (H-Y)-specific T cell receptor-transgenic CD8+ T cells in transplanted male mice in vivo. Taken together these novel findings indicate that an L-type Ca2+ channel plays a significant role in the Ca2+ influx pathways mediating T lymphocyte activation and proliferation in vitro and in vivo. In T lymphocytes, sustained calcium (Ca2+) influx through Ca2+ channels localized in the plasma membrane is critical for T cell activation and proliferation. Previous studies indicated that voltage-dependent Ca2+ channels (VDCCs) play a role in Ca2+ mobilization during T lymphocyte activation. However, the role of VDCCs in otherwise nonexcitable cells is still poorly understood. We used RT-PCR to identify a transcript encoding the pore-forming α1F-subunit of an L-type Ca2+ channel in T lymphocytes. Its identity was confirmed by DNA sequencing. To further investigate the contribution of Ca2+ influx through VDCCs, we assessed the effects of the 1,4-dihydropyridine L-type Ca2+ channel agonist, (+/-) Bay K 8644, and antagonist, nifedipine, on the human Jurkat T cell leukemia line, human peripheral blood T lymphocytes and mouse splenocytes. We found that treatment of T lymphocytes with (+/-) Bay K 8644 increased intracellular Ca2+ and induced the activation of phosphoextracellular-regulated kinase 1/2 (Erk1/2), whereas nifedipine blocked Ca2+ influx, the activity of Erk1/2 and nuclear factor of activated T cells (NFAT), interleukin-2 (IL-2) production, and IL-2 receptor expression. Nifedipine also significantly suppressed splenocyte proliferation in an in vitro mixed lymphocyte reaction and the proliferation of male antigen (H-Y)-specific T cell receptor-transgenic CD8+ T cells in transplanted male mice in vivo. Taken together these novel findings indicate that an L-type Ca2+ channel plays a significant role in the Ca2+ influx pathways mediating T lymphocyte activation and proliferation in vitro and in vivo. In T lymphocytes, calcium (Ca2+) plays a fundamental role as a second messenger in regulating numerous cellular functions, including activation, proliferation, and death (1Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 11-21Crossref PubMed Scopus (4493) Google Scholar, 2Medema J.P. Borst J. Hum. Immunol. 1999; 60: 403-411Crossref PubMed Scopus (22) Google Scholar). The events surrounding Ca2+ mobilization in T lymphocytes are tightly regulated through membrane receptors, signaling molecules, and ion channels. Intracellular Ca2+ release is initiated through the recognition of antigen/major histocompatibility complex by the T cell receptor (TCR) 1The abbreviations used are: TCRT cell receptorIP3inositol 1,4,5-trisphosphateERendoplasmic reticulumNFATnuclear factor of activated T cellsIL-2interleukin-2IP3RIP3 receptorTRPtransient receptor potentialVDCCsvoltage-dependent Ca2+ channelsCRAC channelCa2+ release-activated Ca2+ channelICRACCRAC currentDHP1,4-dihydropyridinePHAphytohemagglutininConAconcanavalin APBMCsperipheral blood mononuclear cellsTgtransgenicmAbmonoclonal antibodyrhIL-2recombinant human IL-2PBTsperipheral blood T lymphocytesMAPmitogen-activated proteinErk1/2extracellular-regulated kinase1/2TPA12-O-tetradecanoylphorbol 13-acetateIL-2RIL-2 receptorPIpropidium iodideCFSE5-(and-6)-carboxyfluorescein diacetate, succinimidyl esterMLRmixed lymphocyte reactionKv channelsvoltage-dependent potassium channelsK+potassiumNTno treatment./CD3 complex during T lymphocyte activation (3van Leeuwen J.E. Samelson L.E. Curr. Opin. Immunol. 1999; 11: 242-248Crossref PubMed Scopus (217) Google Scholar). Following ligation of the TCR, non-receptor tyrosine kinases phosphorylate and activate phospholipase Cγ1, which cleaves phosphatidylinositol 4,5-bisphosphate from plasma membrane phospholipids to generate diacylglycerol and inositol 1,4,5-trisphosphate (IP3) (4Scharenberg A.M. Kinet J.P. Cell. 1998; 94: 5-8Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Elevated levels of IP3 in the cytosol lead to the release of Ca2+ from intracellular stores in the endoplasmic reticulum (ER) and a sustained Ca2+ influx from the extracellular space (5Patel S. Joseph S.K. Thomas A.P. Cell Calcium. 1999; 25: 247-264Crossref PubMed Scopus (373) Google Scholar, 6Guse A.H. Crit. Rev. Immunol. 1998; 18: 419-448Crossref PubMed Google Scholar). A sustained Ca2+ signal ranging from a concentration of ∼200 nm to >1 μm for up to 48 h is necessary to activate nuclear factor of activated T cells (NFAT), a transcription factor that regulates the expression of various cytokine genes including interleukin-2 (IL-2) (7Lewis R.S. Annu. Rev. Immunol. 2001; 19: 497-521Crossref PubMed Scopus (707) Google Scholar). T cell receptor inositol 1,4,5-trisphosphate endoplasmic reticulum nuclear factor of activated T cells interleukin-2 IP3 receptor transient receptor potential voltage-dependent Ca2+ channels Ca2+ release-activated Ca2+ channel CRAC current 1,4-dihydropyridine phytohemagglutinin concanavalin A peripheral blood mononuclear cells transgenic monoclonal antibody recombinant human IL-2 peripheral blood T lymphocytes mitogen-activated protein extracellular-regulated kinase1/2 12-O-tetradecanoylphorbol 13-acetate IL-2 receptor propidium iodide 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester mixed lymphocyte reaction voltage-dependent potassium channels potassium no treatment. Although the mechanisms of Ca2+ release from the intracellular stores within T lymphocytes are well characterized, the Ca2+ entry pathway from extracellular sources into T lymphocytes still remains elusive despite the fact that it contributes to the majority of elevated intracellular Ca2+ during T lymphocyte activation (8Haverstick D.M. Engelhard V.H. Gray L.S. J. Immunol. 1991; 146: 3306-3313PubMed Google Scholar). Several models for Ca2+ channels in the plasma membrane of T lymphocytes have been proposed, including IP3 receptor (IP3R) Ca2+ channels, mammalian homologues of transient receptor potential (TRP) Ca2+ channels, and L-type voltage-dependent Ca2+ channels (VDCCs). Initially, investigators suggested that a plasma membrane IP3R Ca2+ channel, similar to the IP3R found in the ER, was responsible for Ca2+ influx in T lymphocytes (9Jayaraman T. Ondriasova E. Ondrias K. Harnick D.J. Marks A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6007-6011Crossref PubMed Scopus (132) Google Scholar). A recent study confirmed that T lymphocytes express three isoforms of the IP3R Ca2+ channel as integral plasma membrane proteins (10Tanimura A. Tojyo Y. Turner R.J. J. Biol. Chem. 2000; 275: 27488-27493Abstract Full Text Full Text PDF PubMed Google Scholar). However, the IP3R isoforms exhibit functional redundancy; defining the respective contributions of these channels to Ca2+ influx during T cell activation has therefore been difficult (11Hirota J. Baba M. Matsumoto M. Furuichi T. Takatsu K. Mikoshiba K. Biochem. J. 1998; 333: 615-619Crossref PubMed Scopus (29) Google Scholar). Studies based on electrophysiology of T lymphocytes lead to a second model for Ca2+ entry across the plasma membrane through Ca2+-release-activated Ca2+ (CRAC) channels (12Kerschbaum H.H. Cahalan M.D. Science. 1999; 283: 836-839Crossref PubMed Scopus (129) Google Scholar, 13Fomina A.F. Fanger C.M. Kozak J.A. Cahalan M.D. J. Cell Biol. 2000; 150: 1435-1444Crossref PubMed Scopus (90) Google Scholar). Although the molecular identity of the CRAC channel is still unclear, potential gene candidates include the TRP gene superfamily of ion channels (14Gamberucci A. Giurisato E. Pizzo P. Tassi M. Giunti R. McIntosh D.P. Benedetti A. Biochem. J. 2002; 364: 245-254Crossref PubMed Scopus (83) Google Scholar, 15Cui J. Bian J.S. Kagan A. McDonald T.V. J. Biol. Chem. 2002; 277: 47175-47183Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 16Sano Y. Inamura K. Miyake A. Mochizuki S. Yokoi H. Matsushime H. Furuichi K. Science. 2001; 293: 1327-1330Crossref PubMed Scopus (385) Google Scholar). Currently, CaT1 appears to be the primary TRP gene candidate for the CRAC channel, but the CaT1 gene product does not seem to exhibit all of the electrophysiological properties associated with CRAC current (ICRAC) (15Cui J. Bian J.S. Kagan A. McDonald T.V. J. Biol. Chem. 2002; 277: 47175-47183Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 17Yue L. Peng J.B. Hediger M.A. Clapham D.E. Nature. 2001; 410: 705-709Crossref PubMed Scopus (320) Google Scholar). There is also evidence to support the existence of voltage-dependent-like Ca2+ channels in the plasma membrane of T lymphocytes. VDCCs are heteromultimeric proteins whose conformations are sensitive to changes in the electrical potential across the plasma membrane (18Hofmann F. Lacinova L. Klugbauer N. Rev. Physiol. Biochem. Pharmacol. 1999; 139: 33-87Crossref PubMed Google Scholar). The basis of the VDCC model is that nonexcitable cells, such as T lymphocytes, may express a Ca2+ channel that shares common structural features with a VDCC of electrically excitable cells but is not gated by changes in membrane potential. Initial support for the presence of voltage-dependent-like Ca2+ channels in T lymphocytes came when Densmore et al. (19Densmore J.J. Szabo G. Gray L.S. FEBS Lett. 1992; 312: 161-164Crossref PubMed Scopus (22) Google Scholar, 20Densmore J.J. Haverstick D.M. Szabo G. Gray L.S. Am. J. Physiol. 1996; 271: C1494-C1503Crossref PubMed Google Scholar) identified an electrically responsive current in the plasma membrane of Jurkat T lymphocytes. This "voltage-operable" current had different electrophysiological properties than ICRAC, but was activated through the TCR/CD3 complex and Ca2+ store depletion (19Densmore J.J. Szabo G. Gray L.S. FEBS Lett. 1992; 312: 161-164Crossref PubMed Scopus (22) Google Scholar, 20Densmore J.J. Haverstick D.M. Szabo G. Gray L.S. Am. J. Physiol. 1996; 271: C1494-C1503Crossref PubMed Google Scholar). RTPCR analysis has also shown that transcripts of the pore-forming α1C- and α1S-subunits of L-type VDCCs are expressed in Jurkat T cells (21Brereton H.M. Harland M.L. Froscio M. Petronijevic T. Barritt G.J. Cell Calcium. 1997; 22: 39-52Crossref PubMed Scopus (35) Google Scholar). In addition, Savignac et al. (22Savignac M. Badou A. Moreau M. Leclerc C. Guery J.C. Paulet P. Druet P. Ragab-Thomas J. Pelletier L. Faseb J. 2001; 15: 1577-1579Crossref PubMed Scopus (51) Google Scholar) demonstrated that murine T cell hybridomas express L-type Ca2+ channel mRNA and protein. Several pharmacological studies have provided further evidence to support the expression of VDCCs in T lymphocytes. For instance, it has been reported that the synthetic 1,4-dihydropyridine (DHP) L-type Ca2+ channel antagonist, nifedipine, is a potent suppressor of T lymphocyte proliferation. Based upon an in vitro [3H]thymidine uptake assay, Birx et al. (23Birx D.L. Berger M. Fleisher T.A. J. Immunol. 1984; 133: 2904-2909PubMed Google Scholar) demonstrated that 0.001–100 μm nifedipine prevented the proliferation of human T lymphocytes in response to the mitogens, phytohemagglutinin (PHA), and concanavalin A (ConA). In a similar study, human peripheral blood mononuclear cells (PBMCs) stimulated with PHA were unable to proliferate in the presence of 10–200 μm nifedipine; the addition of IL-2 restored the proliferative response in the nifedipine-treated cells (24Gelfand E.W. Cheung R.K. Grinstein S. Mills G.B. Eur. J. Immunol. 1986; 16: 907-912Crossref PubMed Scopus (76) Google Scholar). Furthermore, it has been demonstrated through in vitro proliferation assays that nifedipine has a dose-dependent inhibitory effect on T lymphocyte proliferation when added in combination with the immunosuppressive agent cyclosporin A (25Padberg W.M. Bodewig C. Schafer H. Muhrer K.H. Schwemmle K. Transplant Proc. 1990; 22: 2337PubMed Google Scholar, 26Marx M. Weber M. Merkel F. Meyer zum Buschenfelde K.H. Kohler H. Nephrol. Dial. Transplant. 1990; 5: 1038-1044Crossref PubMed Scopus (35) Google Scholar). The aim of this work was to understand the contribution of Ca2+ influx through VDCCs during T lymphocyte activation and proliferation. We began this investigation by using a PCR assay to demonstrate for the first time that the pore-forming α1F-subunit L-type Ca2+ channel transcript is expressed in human T lymphocytes. After confirming the presence of a VDCC in T lymphocytes, we then determined that L-type Ca2+ channels play a critical role in TCR-induced activation. We show that both (+/-) Bay K 8644 (a DHP agonist that induces L-type Ca2+ channel opening) and nifedipine (a DHP antagonist that blocks L-type Ca2+ channels) can modulate early and late signaling events during T lymphocyte activation and proliferation. The results in this study collectively suggest the presence of a DHP-sensitive L-type VDCC in the plasma membrane of T lymphocytes. Mice—C57Bl/6 female mice bearing a transgenic (Tg) TCRαβ receptor specific for the male antigen H-Y were provided by Dr. Philippe Poussier at Sunnybrook and Women's College, Health Sciences Centre, Toronto, Canada. Balb/c and C57Bl/6 mice (Charles River Laboratories, Wilmington, MA) were housed in the animal facilities at University of British Columbia and were used between 8 and 12 weeks of age. All mice studies were approved by the Committee on Animal Care at the University of British Columbia using the guidelines set out by the Canadian Council on Animal Care. Cell Line and Culture Conditions—The human T cell leukemia line Jurkat clone E6–1 was obtained from American Type Culture Collection (ATCC, Manassas, VA) maintained in RPMI 1640 medium supplemented with 10% FBS, 2 mm glutamine, 20 mm HEPES, and 1 mm sodium pyruvate. Isolation and Culture of Human Peripheral Blood T Lymphocytes— Whole blood (10–50 ml) was collected from healthy human male and female donors (n = 15). PBMCs were separated by centrifugation at 900 × g for 30 min at 18–20 °C over a Ficoll-Paque PLUS (Amersham Biosciences) gradient. The resulting PBMC layer was washed and resuspended in RPMI supplemented with 10% FBS, 2 mm glutamine, 20 mm HEPES, and 1 mm sodium pyruvate. PBMCs were then stimulated for 24 h with 10 μg/ml plate-bound anti-CD3 monoclonal antibody (mAb), OKT3 (ATCC), and resuspended in RPMI 1640 containing 5 ng/ml recombinant human IL-2 (rhIL-2) (Serologicals Corporation, Norcross, GA). After growing for 7 d in rhIL-2, the purity of the human peripheral blood T lymphocytes (PBTs) was analyzed on a FACSCalibur cytometer (BD Biosciences) with FITC-conjugated OKT3, FITC-conjugated mouse anti-IgG2a isotype control (Caltag, Burlingame, CA), Cyanine-5-conjugated anti-CD4 mAb (BD Pharmingen), PE-conjugated anti-CD8 mAb (BD Pharmingen), APC-conjugated anti-CD14 mAb (BD Pharmingen), FITC-conjugated anti-CD15 mAb (BD Pharmingen), and PE-conjugated anti-CD19 mAb (BD Pharmingen). Experiments with the activated human PBTs were conducted with cells from day 8–14 in culture. Nested RT-PCR and DNA Sequencing—First strand cDNAs were synthesized with an oligo(dT) primer using 1 μg of total RNA extracted from Weri-Rb1 retinoblastoma, Jurkat T cells, PBTs, CD4+ T cells, and CD8+ T cells with the RNeasy Kit (Qiagen, Mississauga, Ontario). MACS CD4 and CD8 microbeads (Miltenyi Biotec, Auburn, CA) were used to positively select and separate human CD4+ and CD8+ T cells from PBTs, respectively. Marathon-ready human retina and human spleen cDNA were purchased from Clontech and FirstChoice PCR-Ready human liver cDNA was from Ambion (Austin, TX). RT-PCR fragments (∼770 bp) spanning exons 28–35 of the L-type Ca2+ channel α1F-subunit gene, CACNA1F, were generated with sense primer (5′-GGACCATGGCCCCATCTATAATTACCG-3′) and antisense primer (5′-CCTGAAGAGCCACCTTGCCGAAC-3′). For nested amplification of the CACNA1F gene, PCR fragments (∼180 bp) spanning exons 29 to 30 were generated with sense primer (5′-GAACCCGCATCAGTATCGTG-3′) and antisense primer (5′-AATAGTGAAGAGGCCAGTGAAGACC-3′). The housekeeping gene, rig/S15, which encodes a small ribosomal subunit protein, was amplified with sense primer (5′-TTCCGCAAGTTCACCTACC-3′) and antisense primer (5′-CGGGCCGGCCATGCTTTACG-3′) (27Kitagawa M. Takasawa S. Kikuchi N. Itoh T. Teraoka H. Yamamoto H. Okamoto H. FEBS Lett. 1991; 283: 210-214Crossref PubMed Scopus (47) Google Scholar). RT-PCR and nested PCR reactions were performed with Platinum Taq polymerase (Invitrogen) and were conducted in a Whatman Biometra UnoII Thermocycler at 94 °C for 1 min, then 30 cycles of 94 °C for 30 s, 60 °C for 30 s and 72 °C for 1 min, followed by a 10-min extension at 72 °C. PCR fragments were resolved on a 1% agarose gel and visualized by staining with ethidium bromide. The resulting 178 bp fragments were subcloned into pCR2.1-TOPO vector (Invitrogen) and the nucleotide sequence determined using standard m13R primer at the DNA Sequencing CORE Facility, University of Florida. Measurement of Intracellular Ca2+ Levels—Intracellular Ca2+ levels were measured using the ratiometric Ca2+ indicator indo-1 acetoxymethyl ester dye (Molecular Probes, Eugene, OR) according to manufacturer's recommendations. In brief, Jurkat T cells or human PBTs (donors, n = 3) at 1 × 107 cells/ml were loaded with 1 μm indo-1 for 1 h at 37 °C in MEM. For analysis, 100 μl of cell suspension (1 × 106 cells) was added to either 1.9 ml of MEM or Ca2+-free S-MEM. Indo-1 loaded T cells were then examined for 10 min time periods following induction at the 2 min mark with either 10–100 μm (+/-) Bay K 8644 (Calbiochem), 2 μm ionomycin (Calbiochem) or Me2SO solvent using a FACS-Vantage S.E. (BD Biosciences). Jurkat T cells and human PBTs loaded with indo-1 were also preincubated with 1–200 μm nifedipine (Calbiochem) or Me2SO with or without extracellular Ca2+ in the medium for 10 min. At the 2-min mark, Jurkat T cells were stimulated with 10 μg/ml soluble OKT3, whereas human PBTs required a combination of 2 μg/ml soluble anti-CD28 mAb (Sigma), 10 μg/ml soluble OKT3 and 40 μg/ml soluble rabbit anti-mouse IgG polyclonal Ab (Sigma), which served as a cross-linking Ab, to activate Ca2+ influx. Anti-CD3 stimulation using the OKT3 mAb alone did not activate Ca2+ influx in PBTs. The change in intracellular Ca2+ concentration was determined through the ratio of emission signals of indo-1 at 405 nm and 485 nm, representing the ratio of Ca2+-bound to Ca2+-free indo-1, respectively. In all experiments, the amount of Me2SO solvent was equal to or less than 0.5% of the total treatment volume. (+/-) Bay K 8644 and nifedipine were both prepared in the dark as a stock solution of 200 mm dissolved in Me2SO. Immunoblot Analysis of Phospho-p44/42 MAP Kinase—Jurkat T cells or human PBTs were washed, resuspended at 1 × 107 cells/ml in RPMI 1640 and incubated for4hat37 °C. Cells were then preincubated with or without 2 mm EGTA for 15 min to chelate Ca2+, followed by 10 min stimulation with either (+/-) Bay K 8644 or 2 μm ionomycin at 37 °C. Jurkat T cells were also preincubated with either Me2SO, 100 μm or 200 μm nifedipine for 1 h, followed by 10 min stimulation with 100 μm (+/-) Bay K 8644 at 37 °C. Additionally, as a positive control for phospho-p44/42 MAP kinase activation, Jurkat T cells were stimulated with 10 μg/ml soluble OKT3 for 10 min at 37 °C. Following stimulation, cells were lysed in 200 μl of lysis buffer containing 50 mm Tris-HCl (pH 7.5), 150 mm NaCl, 10% glycerol, 1% Nonidet P-40, 5 mm EDTA, 1 mm sodium vandadate, 5 mm sodium fluoride, 1 mm sodium molybdate, 5 mm β glycerol phosphate, in the presence of 10 μg/ml soybean trypsin inhibitor, pepstatin, and 40 μg/ml phenylmethylsulfonyl fluoride. Cell lysates were denatured by boiling in SDS sample buffer, run on 12% SDS-PAGE gel and transferred to nitrocellulose membrane. Western blot analysis was performed with phospho-p44/42 MAP kinase rabbit polyclonal Ab (Cell Signaling Technology, Beverly, MA). After development, the blots were stripped in 62.5 mm Tris-HCl (pH 7.5), 0.2% SDS, and 100 mm 2-mercaptoethanol for 30 min at 50 °C and then reprobed with extracellular regulated kinase1/2 (Erk1/2) (K-23) polyclonal Ab (Santa Cruz Biotechnology, Santa Cruz, CA) as a protein-loading control. NFAT Luciferase Assay—1 × 107 Jurkat T cells were washed and resuspended in Opti-MEM. Cells were incubated with either 20 μg of pNFAT-TA-Luc or pTA-Luc (Clontech) for 5 min at 4 °C and transfected by electroporation using a BioRad Gene Pulser Electroporator set at 280 V, 975 μF. 40–48 h after transfection, cells at 1 × 106 cells/ml were incubated with nifedipine (1–200 μm) or Me2SO for 1 h at 37 °C, followed by stimulation with 10 μg/ml soluble OKT3 for 6 h at 37 °C. NFAT-dependent luciferase activity was assayed on 1 × 105 cells/100 μl using the procedures outlined in the Bright-Glo Luciferase Assay System (Promega, Madison, WI). Luciferase activity was measured in a microplate luminometer. IL-2 Assay and IL-2 Receptor Expression—Jurkat T cells or human PBTs at 1 × 106 cells/ml were incubated with Me2SO or nifedipine (1–200 μm) for 1 h at 37 °C. Cells were then transferred to a 24-well plate immobilized with 10 μg/ml OKT3, 10 nm 12-O-tetradecanoylphorbol 13-acetate (TPA) (Sigma) was added, and cells were incubated at 37 °C. After 24 h, supernatants were quantified for IL-2 concentration by a standard sandwich ELISA technique (R&D Systems, Minneapolis, MN). To determine whether the Ca2+ ionophore, ionomycin, could reverse the inhibitory effect of nifedipine, Jurkat T cells or human PBTs at 1 × 106 cells/ml were incubated with either Me2SO or 1–50 μm nifedipine for 1 h. Cells were then stimulated for 24 h with 10 μg/ml plate-bound OKT3, 10 nm TPA and, where appropriate, 2 μm ionomycin. The concentration of IL-2 in the supernatants was quantified by sandwich ELISA. IL-2 receptor (IL-2R) expression was determined through flow cytometry by directly staining cells with human IL-2Rα mAb, clone 7G7/B6 (Upstate Biotechnology, Lake Placid, NY) and FITC-conjugated goat anti-mouse IgG Ab (Jackson ImmunoResearch, West Grove, PA). Cell viability was assessed by staining dead cells with 2 μg/ml propidium iodide (PI) (Sigma). Mixed Lymphocyte Reaction—Splenocytes from C57Bl/6 (H-2b) mice at 2 × 106 cells were incubated with nifedipine for 1 h at 37 °C. C57Bl/6 splenocytes were then stimulated with 2000R-irradiated stimulator splenocytes at 4 × 106 cells from allogeneic Balb/c (H-2d) or syngeneic C57Bl/6 (H-2b) mice for 5 to 6 d at 37 °C with nifedipine remaining in the culture medium. Splenocytes were grown in RPMI 1640 supplemented with 10% FBS, 2 mm glutamine, 50 nm 2-mercaptoethanol, and 100 units/ml each of penicillin and streptomycin. Proliferation was evaluated by using a flow cytometer-based bead assay as previously described (28Carlow D.A. Corbel S.Y. Ziltener H.J. J. Immunol. 2001; 166: 256-261Crossref PubMed Scopus (32) Google Scholar). In Vivo Proliferation Assay—Thymocytes from C57Bl/6 female mice bearing a Tg TCRαβ receptor specific for the male antigen were loaded with 5 μm 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester (CFSE) (Molecular Probes) for 7 min at room temperature. 20–30 × 106 CFSE loaded Tg thymocytes were i.v. injected into the tail vein of female or male C57Bl/6 recipients, followed by one intraperitoneal injection of either vehicle control or 15 mg/kg nifedipine into the C57Bl/6 males 20–24 h later. Nifedipine was prepared as a 1 mg/ml stock solution, dissolved in PBS containing 5% ethanol and 1% Tween-80. 40 h after the initial i.v. injection, spleens were harvested and single cell suspensions were prepared. For cell staining, splenocytes were suspended in Dulbecco's modified Eagle's medium and labeled with PE-conjugated anti-CD8α mAb (BD Pharmingen) and biotin-conjugated anti-TCRα mAb, clone T3.70 (provided by Dr. Hung-sia Teh, UBC, Vancouver, Canada), which is specific for Tg TCRα. Cells were then stained with Cychrome conjugated-streptavidin, washed, and analyzed on a FACSCalibur cytometer (BD Biosciences). Proliferation of female H-Y-specific TCRαβ receptor CD8+ T cells in vivo was quantified by determining the percent total of gated viable, CFSE+, CD8+, and Tg TCRhigh cells in successive cell divisions using CellQuest software (BD Biosciences). As a result of CFSE labeling being distributed equally between daughter cells, a halving of cellular fluorescence intensity marked each successive cell division among proliferating cells. Statistical Analysis—Statistical significance was determined by the ANOVA test, using two-factorial design without replication. For all tests, p < 0.01 was considered to indicate statistical significance. All standard errors shown represent the S.D. L-Type Ca2+ Channel Transcript Is Expressed in T Lymphocytes—Nifedipine has been shown to antagonize Ca2+ influx through l-type Ca2+ channels. Since previous studies reported that nifedipine interfered with T lymphocyte proliferation we sought to demonstrate the expression of an L-type Ca2+ channel in T lymphocytes. Using a nested RT-PCR based assay a PCR product specific to the pore-forming α1F-subunit of an L-type VDCC was amplified from T lymphocytes (Fig. 1A). Human retina and Weri-Rb1 retinoblastoma cDNAs were used as positive controls for the PCR assay since the α1F-subunit gene, CACNA1F, was first cloned from human retina and is highly expressed in this tissue (29Strom T.M. Nyakatura G. Apfelstedt-Sylla E. Hellebrand H. Lorenz B. Weber B.H. Wutz K. Gutwillinger N. Ruther K. Drescher B. Sauer C. Zrenner E. Meitinger T. Rosenthal A. Meindl A. Nat. Genet. 1998; 19: 260-263Crossref PubMed Scopus (395) Google Scholar, 30Bech-Hansen N.T. Naylor M.J. Maybaum T.A. Pearce W.G. Koop B. Fishman G.A. Mets M. Musarella M.A. Boycott K.M. Nat. Genet. 1998; 19: 264-267Crossref PubMed Scopus (432) Google Scholar). In the initial studies examining α1F-subunit gene expression, the α1F-subunit mRNA was not detected in lymphoblastoid tissue (29Strom T.M. Nyakatura G. Apfelstedt-Sylla E. Hellebrand H. Lorenz B. Weber B.H. Wutz K. Gutwillinger N. Ruther K. Drescher B. Sauer C. Zrenner E. Meitinger T. Rosenthal A. Meindl A. Nat. Genet. 1998; 19: 260-263Crossref PubMed Scopus (395) Google Scholar). The expression may have been overlooked by the lack of a nested RT-PCR-based assay. Using nucleotide sequencing, we were able to confirm that the ∼180 bp amplified PCR product from Jurkat T cells, human spleen, PBTs, CD4+, and CD8+ T lymphocytes shares 100% nucleotide identity to the L-type Ca2+ channel α1F-subunit gene expressed in human retina and Weri-Rb1 retinoblastoma (Fig. 1B). The α1F-subunit is not expressed ubiquitously in human cells since we were not able to detect α1F-subunit expression in normal human liver. Induction of Ca2+ Influx in Jurkat T Cell Leukemia Line and Human PBTs by L-Type Ca2+ Channel Agonist, (+/-) Bay K 8644 —To demonstrate that L-type Ca2+ channels contribute to Ca2+ entry, we tested the effect of the DHP derivative, (+/-) Bay K 8644, on Ca2+ influx in human T lymphocytes. It has previously been reported that the treatment of Jurkat T cells with Bay K 8644 in the range of 0.01–100 μm induces a small rise in intracellular Ca2+, indicating the presence of a DHP-sensitive Ca2+ influx pathway in these cells (31Young W. Chen J. Jung F. Gardner P. Mol. Pharmacol. 1988; 34: 239-244PubMed Google Scholar). However, the report did not specify which enantiomer of Bay K 8644 was used. Since levo- and dextro-rotatory enantiomers of Bay K 8644 can induce opposing L-type VDCC activity, the experiment was repeated in this study with (+/-) Bay K 8644, a racemic mixture that has the net effect of enhancing Ca2+ influx through L-type Ca2+ channels (32Barger S.W. Neuroscience. 1999; 89: 101-108Crossref PubMed Scopus (13) Google Scholar). Additionally, we wanted to directly compare the effects of (+/-) Bay K 8644 treatment on Ca2+ influx in Jurkat T cells to the untransformed PBTs since this has not been previously examined. When indo-1-loaded Jurkat T cells and human PBTs were treated with (+/-) Bay K 8644, a dose-dependent increase in the mean ratio of indo-1 bound to Ca2+ (405 nm)/free indo-1 (485 nm) was observed indicating an increase in intracellular Ca2+ (Fig. 2). In both Jurkat T cells and PBTs, 10 μm (+/-) Bay K 8644 induced a small, sustained rise in intracellular Ca2+. However, treatment of Jurkat T cells and PBTs with higher concentrations of (+/-) Bay K 8644 induced different responses in Ca2+ influx. In Jurkat T cells, 50 and 100 μm (+/-) Bay K 8644 induced a sustained increase in cytosolic Ca2+ (Fig. 2A). Interestingly, treatment of human PBTs with either 50 or 100 μm (+/-) Bay K 8644 caused a transient Ca2+ influx that rapidly declined to below
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