Identification and Functional Characterization of Voltage-dependent Calcium Channels in T Lymphocytes*

In T lymphocytes, sustained calcium (Ca 2 (cid:1) ) influx through Ca 2 (cid:1) channels localized in the plasma membrane is critical for T cell activation and proliferation. Previous studies indicated that voltage-dependent Ca 2 (cid:1) channels (VDCCs) play a role in Ca 2 (cid:1) mobilization during T lymphocyte activation. However, the role of VDCCs in otherwise nonexcitable cells is still poorly under-stood. We used RT-PCR to identify a transcript encoding the pore-forming (cid:2) 1F -subunit of an L-type Ca 2 (cid:1) channel in T lymphocytes. Its identity was confirmed by DNA sequencing. To further investigate the contribution of Ca 2 (cid:1) influx through VDCCs, we assessed the effects of the 1,4-dihydropyridine L-type Ca 2 (cid:1) channel agonist, ( (cid:1) / (cid:3) ) 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 ( (cid:1) / (cid:3) ) Bay K 8644 increased intracellular Ca 2 (cid:1) and induced the activation of phosphoextracellular-regulated kinase 1/2

In T lymphocytes, calcium (Ca 2ϩ ) plays a fundamental role as a second messenger in regulating numerous cellular functions, including activation, proliferation, and death (1,2). The events surrounding Ca 2ϩ mobilization in T lymphocytes are tightly regulated through membrane receptors, signaling molecules, and ion channels. Intracellular Ca 2ϩ release is initiated through the recognition of antigen/major histocompatibility complex by the T cell receptor (TCR) 1 /CD3 complex during T lymphocyte activation (3). Following ligation of the TCR, nonreceptor tyrosine kinases phosphorylate and activate phospholipase C␥1, which cleaves phosphatidylinositol 4,5-bisphosphate from plasma membrane phospholipids to generate diacylglycerol and inositol 1,4,5-trisphosphate (IP 3 ) (4). Elevated levels of IP 3 in the cytosol lead to the release of Ca 2ϩ from intracellular stores in the endoplasmic reticulum (ER) and a sustained Ca 2ϩ influx from the extracellular space (5,6). A sustained Ca 2ϩ signal ranging from a concentration of ϳ200 nM to Ͼ1 M 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) (7).
Although the mechanisms of Ca 2ϩ release from the intracellular stores within T lymphocytes are well characterized, the Ca 2ϩ entry pathway from extracellular sources into T lymphocytes still remains elusive despite the fact that it contributes to the majority of elevated intracellular Ca 2ϩ during T lymphocyte activation (8). Several models for Ca 2ϩ channels in the plasma membrane of T lymphocytes have been proposed, including IP 3 receptor (IP 3 R) Ca 2ϩ channels, mammalian homologues of transient receptor potential (TRP) Ca 2ϩ channels, and L-type voltage-dependent Ca 2ϩ channels (VDCCs). Initially, investigators suggested that a plasma membrane IP 3 R Ca 2ϩ channel, similar to the IP 3 R found in the ER, was responsible for Ca 2ϩ influx in T lymphocytes (9). A recent study confirmed that T lymphocytes express three isoforms of the IP 3 R Ca 2ϩ channel as integral plasma membrane proteins (10). However, the IP 3 R isoforms exhibit functional redundancy; defining the respective contributions of these channels to Ca 2ϩ influx during T cell activation has therefore been difficult (11). Studies based on electrophysiology of T lymphocytes lead to a second model for Ca 2ϩ entry across the plasma membrane through Ca 2ϩ -release-activated Ca 2ϩ (CRAC) channels (12,13). Although the molecular identity of the CRAC channel is still unclear, potential gene candidates include the TRP gene superfamily of ion channels (14 -16). 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 (I CRAC ) (15,17).
There is also evidence to support the existence of voltage-dependent-like Ca 2ϩ 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 (18). The basis of the VDCC model is that nonexcitable cells, such as T lymphocytes, may express a Ca 2ϩ 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 Ca 2ϩ channels in T lymphocytes came when Densmore et al. (19,20) identified an electrically responsive current in the plasma membrane of Jurkat T lymphocytes. This "voltage-operable" current had different electrophysiological properties than I CRAC , but was activated through the TCR/CD3 complex and Ca 2ϩ store depletion (19,20). RT-PCR analysis has also shown that transcripts of the poreforming ␣ 1C -and ␣ 1S -subunits of L-type VDCCs are expressed in Jurkat T cells (21). In addition, Savignac et al. (22) demonstrated that murine T cell hybridomas express L-type Ca 2ϩ 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 Ca 2ϩ channel antagonist, nifedipine, is a potent suppressor of T lymphocyte proliferation. Based upon an in vitro [ 3 H]thymidine uptake assay, Birx et al. (23) demonstrated that 0.001-100 M 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 (PB-MCs) stimulated with PHA were unable to proliferate in the presence of 10 -200 M nifedipine; the addition of IL-2 restored the proliferative response in the nifedipine-treated cells (24). 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 (25,26).
The aim of this work was to understand the contribution of Ca 2ϩ 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 ␣ 1F -subunit L-type Ca 2ϩ channel transcript is expressed in human T lymphocytes. After confirming the presence of a VDCC in T lymphocytes, we then determined that L-type Ca 2ϩ channels play a critical role in TCR-induced activation. We show that both (ϩ/Ϫ) Bay K 8644 (a DHP agonist that induces L-type Ca 2ϩ channel opening) and nifedipine (a DHP antagonist that blocks L-type Ca 2ϩ 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.

EXPERIMENTAL PROCEDURES
Mice-C57Bl/6 female mice bearing a transgenic (Tg) TCR␣␤ 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.
Measurement of Intracellular Ca 2ϩ Levels-Intracellular Ca 2ϩ levels were measured using the ratiometric Ca 2ϩ 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 ϭ 3) at 1 ϫ 10 7 cells/ml were loaded with 1 M indo-1 for 1 h at 37°C in MEM. For analysis, 100 l of cell suspension (1 ϫ 10 6 cells) was added to either 1.9 ml of MEM or Ca 2ϩ -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 M (ϩ/Ϫ) Bay K 8644 (Calbiochem), 2 M ionomycin (Calbiochem) or Me 2 SO 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 M nifedipine (Calbiochem) or Me 2 SO with or without extracellular Ca 2ϩ in the medium for 10 min. At the 2-min mark, Jurkat T cells were stimulated with 10 g/ml soluble OKT3, whereas human PBTs required a combination of 2 g/ml soluble anti-CD28 mAb (Sigma), 10 g/ml soluble OKT3 and 40 g/ml soluble rabbit anti-mouse IgG polyclonal Ab (Sigma), which served as a cross-linking Ab, to activate Ca 2ϩ influx. Anti-CD3 stimulation using the OKT3 mAb alone did not activate Ca 2ϩ influx in PBTs. The change in intracellular Ca 2ϩ concentration was determined through the ratio of emission signals of indo-1 at 405 nm and 485 nm, representing the ratio of Ca 2ϩbound to Ca 2ϩ -free indo-1, respectively. In all experiments, the amount of Me 2 SO solvent was equal to or less than 0.5% of the total treatment volume. (ϩ/Ϫ) Bay K 8644 and nifedipine were both prepared in the dark as a stock solution of 200 mM dissolved in Me 2 SO.
Immunoblot Analysis of Phospho-p44/42 MAP Kinase-Jurkat T cells or human PBTs were washed, resuspended at 1 ϫ 10 7 cells/ml in RPMI 1640 and incubated for 4 h at 37°C. Cells were then preincubated with or without 2 mM EGTA for 15 min to chelate Ca 2ϩ , followed by 10 min stimulation with either (ϩ/Ϫ) Bay K 8644 or 2 M ionomycin at 37°C. Jurkat T cells were also preincubated with either Me 2 SO, 100 M or 200 M nifedipine for 1 h, followed by 10 min stimulation with 100 M (ϩ/Ϫ) 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 g/ml soluble OKT3 for 10 min at 37°C. Following stimulation, cells were lysed in 200 l 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 ␤ glycerol phosphate, in the presence of 10 g/ml soybean trypsin inhibitor, pepstatin, and 40 g/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 ϫ 10 7 Jurkat T cells were washed and resuspended in Opti-MEM. Cells were incubated with either 20 g 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 F. 40 -48 h after transfection, cells at 1 ϫ 10 6 cells/ml were incubated with nifedipine (1-200 M) or Me 2 SO for 1 h at 37°C, followed by stimulation with 10 g/ml soluble OKT3 for 6 h at 37°C. NFAT-dependent luciferase activity was assayed on 1 ϫ 10 5 cells/100 l 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 ϫ 10 6 cells/ml were incubated with Me 2 SO or nifedipine (1-200 M) for 1 h at 37°C. Cells were then transferred to a 24-well plate immobilized with 10 g/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 Ca 2ϩ ionophore, ionomycin, could reverse the inhibitory effect of nifedipine, Jurkat T cells or human PBTs at 1 ϫ 10 6 cells/ml were incubated with either Me 2 SO or 1-50 M nifedipine for 1 h. Cells were then stimulated for 24 h with 10 g/ml plate-bound OKT3, 10 nM TPA and, where appropriate, 2 M ionomycin. The concentration of IL-2 in the supernatants was quantified by sandwich ELISA.
In Vivo Proliferation Assay-Thymocytes from C57Bl/6 female mice bearing a Tg TCR␣␤ receptor specific for the male antigen were loaded with 5 M 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester (CFSE) (Molecular Probes) for 7 min at room temperature. 20 -30 ϫ 10 6 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-CD8␣ mAb (BD Pharmingen) and biotin-conjugated anti-TCR␣ mAb, clone T3.70 (provided by Dr. Hung-sia Teh, UBC, Vancouver, Canada), which is specific for Tg TCR␣. Cells were then stained with Cychrome conjugated-streptavidin, washed, and analyzed on a FACSCalibur cytometer (BD Biosciences). Proliferation of female H-Y-specific TCR␣␤ receptor CD8 ϩ T cells in vivo was quantified by determining the percent total of gated viable, CFSE ϩ , CD8 ϩ , and Tg TCR high 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 Ͻ 0.01 was considered to indicate statistical significance. All standard errors shown represent the S.D.

L-Type Ca 2ϩ Channel Transcript Is Expressed in T Lympho
cytes-Nifedipine has been shown to antagonize Ca 2ϩ influx through L-type Ca 2ϩ channels. Since previous studies reported that nifedipine interfered with T lymphocyte proliferation we sought to demonstrate the expression of an L-type Ca 2ϩ channel in T lymphocytes. Using a nested RT-PCR based assay a PCR product specific to the pore-forming ␣ 1F -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 ␣ 1F -subunit gene, CACNA1F, was first cloned from human retina and is highly expressed in this tissue (29,30). In the initial studies examining ␣ 1F -subunit gene expression, the ␣ 1F -subunit mRNA was not detected in lymphoblastoid tissue (29). 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 ϳ180 bp amplified PCR product from Jurkat T cells, human spleen, PBTs, CD4 ϩ , and CD8 ϩ T lymphocytes shares 100% nucleotide identity to the L-type Ca 2ϩ channel ␣ 1F -subunit gene expressed in human retina and Weri-Rb1 retinoblastoma (Fig. 1B). The ␣ 1F -subunit is not expressed ubiquitously in human cells since we were not able to detect ␣ 1F -subunit expression in normal human liver.
Induction of Ca 2ϩ Influx in Jurkat T Cell Leukemia Line and Human PBTs by L-Type Ca 2ϩ Channel Agonist, (ϩ/Ϫ) Bay K 8644 -To demonstrate that L-type Ca 2ϩ channels contribute to Ca 2ϩ entry, we tested the effect of the DHP derivative, (ϩ/Ϫ) Bay K 8644, on Ca 2ϩ 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 M induces a small rise in intracellular Ca 2ϩ , indicating the presence of a DHPsensitive Ca 2ϩ influx pathway in these cells (31). 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 (ϩ/Ϫ) Bay K 8644, a racemic mixture that has the net effect of enhancing Ca 2ϩ influx through L-type Ca 2ϩ channels (32). Additionally, we wanted to directly compare the effects of (ϩ/Ϫ) Bay K 8644 treatment on Ca 2ϩ 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 (ϩ/Ϫ) Bay K 8644, a dose-dependent increase in the mean ratio of indo-1 bound to Ca 2ϩ (405 nm)/free indo-1 (485 nm) was observed indicating an increase in intracellular Ca 2ϩ (Fig. 2). In both Jurkat T cells and PBTs, 10 M (ϩ/Ϫ) Bay K 8644 induced a small, sustained rise in intracellular Ca 2ϩ . However, treatment of Jurkat T cells and PBTs with higher concentrations of (ϩ/Ϫ) Bay K 8644 induced different responses in Ca 2ϩ influx. In Jurkat T cells, 50 and 100 M (ϩ/Ϫ) Bay K 8644 induced a sustained increase in cytosolic Ca 2ϩ (Fig. 2A). Interestingly, treatment of human PBTs with either 50 or 100 M (ϩ/Ϫ) Bay K 8644 caused a transient Ca 2ϩ influx that rapidly declined to below baseline after the 10 min time period (Fig. 2B). In the absence of extracellular Ca 2ϩ in the medium, 100 M (ϩ/Ϫ) Bay K 8644 did not cause a rise in intracellular Ca 2ϩ in either Jurkat T cells or human PBTs, indicating that (ϩ/Ϫ) Bay K 8644 treatment with extracellular Ca 2ϩ specifically allowed Ca 2ϩ entrance into these cells. Furthermore, the Me 2 SO solvent alone did not induce significant Ca 2ϩ entry into T lymphocytes. We also examined the effects of the well-characterized lipophilic Ca 2ϩ ionophore, ionomycin, to ensure efficient loading of the indo-1 dye into T lymphocytes. The addition 2 M ionomycin induced a rapid and sustained Ca 2ϩ influx in both Jurkat T cells and human PBTs (Fig. 2, C and D).
L-type Ca 2ϩ Channel Antagonist, Nifedipine Inhibits Anti-CD3-induced Ca 2ϩ Influx in Jurkat T Cell Leukemia Line and Human PBTs-Nifedipine is a blocker of L-type Ca 2ϩ channels and was used to further investigate the role of a DHP sensitive Ca 2ϩ channel in T lymphocytes. While some of the previous reports on nifedipine have examined the nonspecific stimulation of T lymphocytes using the mitogenic lectins PHA and ConA, we focused on the specific activation of T lymphocytes through the TCR/CD3 complex using the anti-CD3 mAb, OKT3. Pretreatment of indo-1-loaded Jurkat T cells and human PBTs with nifedipine in the presence of extracellular Ca 2ϩ resulted in a dose-dependent decrease in the mean ratio of indo-1 bound to Ca 2ϩ (405 nm) versus free indo-1 (485 nm) following anti-CD3 stimulation (Fig. 3, A and B). A decrease in the mean indo-1 ratio demonstrates that nifedipine consistently inhibited anti-CD3-induced Ca 2ϩ influx in both Jurkat T cells and PBTs whereas the Me 2 SO solvent control had no effect.
Although nifedipine clearly inhibited Ca 2ϩ influx in the cells tested, we wanted to determine whether this inhibition was a partial or complete blockage of Ca 2ϩ influx. This would help distinguish whether L-type Ca 2ϩ channels are the only channels that mediate Ca 2ϩ influx or if other channels contribute to the Ca 2ϩ response during T lymphocyte activation. To address this question, we compared nifedipine-inhibited Ca 2ϩ influx with extracellular Ca 2ϩ to Ca 2ϩ influx induced in the absence of extracellular Ca 2ϩ . In both anti-CD3 stimulated Jurkat T cells and PBTs, a rapid, transient increase in intracellular Ca 2ϩ , arising from intracellular Ca 2ϩ stores, was observed when extracellular Ca 2ϩ was absent from the medium (Fig. 3,  C and D, Control, red line). This transient Ca 2ϩ spike was not observed in Jurkat T cells treated with nifedipine in the presence of extracellular Ca 2ϩ , indicating that nifedipine only partially blocked Ca 2ϩ influx in these cells. However, in human PBTs, higher concentrations of nifedipine completely abolished Ca 2ϩ influx since the Ca 2ϩ trace with no extracellular Ca 2ϩ FIG. 1. L-type Ca 2؉ channel is expressed in T lymphocytes. A, using a nested RT-PCR reaction, a ϳ180-bp PCR product corresponding to the channel-forming ␣ 1F -subunit of an L-type VDCC was isolated from human cell lines and tissues, including retina, Weri-Rb1 retinoblastoma, spleen, Jurkat T cells, PBTs, CD4 ϩ , and CD8 ϩ T cells, but was not expressed in normal human liver (top panel). The S15 ribosomal subunit PCR served as a loading control (bottom panel). PCR products were resolved on a 1% agarose gel and visualized by staining with ethidium bromide. B, nucleotide sequence alignment of the ϳ180-bp PCR product amplified from retina, Weri-Rb1 retinoblastoma and T lymphocytes using the European Bioinformatics Institute ClustalW alignment program. The PCR product from human T lymphocytes shares 100% nucleotide identity to the L-type ␣ 1F -subunit VDCC isolated from human retina.
was very similar to PBTs treated with 50 -200 M nifedipine when extracellular Ca 2ϩ was present. We also determined whether nifedipine blocked Ca 2ϩ efflux from intracellular Ca 2ϩ stores. To investigate this, Jurkat T cells and human PBTs were treated with nifedipine and anti-CD3 stimulated in the absence of extracellular Ca 2ϩ (Fig. 3, C and D). Nifedipine treatment did not significantly effect the transient rise in cytosolic Ca 2ϩ from intracellular Ca 2ϩ stores in Jurkat T cells, whereas in human PBTs 50 -200 M nifedipine reduced the efflux of Ca 2ϩ from intracellular stores in these cells.
It should be noted that (ϩ/Ϫ) Bay K 8644 and nifedipine are typically used at 1-300 M on both electrically excitable and nonexcitable cell types. The concentration of DHPs used in these experiments therefore replicated the concentration range of Ca 2ϩ channel modulators applied in other studies (32,33).
(ϩ/Ϫ) Bay K 8644 and Nifedipine Modulate Phospho-p44/42 MAP Kinase Activation in Jurkat T Cells and Human PBTs-The next step in our investigation was to determine whether the Ca 2ϩ influx through L-type Ca 2ϩ channels could activate downstream, Ca 2ϩ -dependent signaling pathways involved in T lymphocyte activation. We examined the expression of the p44/42 MAP kinase, also known as Erk1/2 since a rise in intracellular Ca 2ϩ through Ca 2ϩ ionophores, such as ionomycin and A23187, can induce the activation of Erk1/2 in T lympho-cytes (34). The addition of the specific Ca 2ϩ chelator, EGTA, prior to stimulation of Jurkat T cells, and human PBTs with ionomycin blocks activation of Erk1/2 demonstrating that one mode of Erk1/2 activation in T lymphocytes is through an increase in intracellular Ca 2ϩ (34). Additionally, DHP derivatives are reported to modulate the MAP kinase pathway in neurons, but this phenomenon has not been examined in T lymphocytes (35).
In Jurkat T cells, 50 and 100 M (ϩ/Ϫ) Bay K 8644 stimulation resulted in a rapid and transient phosphorylation of both Erk1 and 2, which is similar to the level of Erk1/2 activated with 2 M ionomycin (Fig. 4A). The activation of Erk1/2 with (ϩ/Ϫ) Bay K 8644 was blocked by pretreatment with EGTA. In human PBTs, (ϩ/Ϫ) Bay K 8644 did not activate Erk1 and only weakly activated Erk2 compared with the ionomycin control (Fig. 4B). The activation of Erk2 with (ϩ/Ϫ) Bay K 8644 was not blocked by pretreatment with EGTA. The treatment of Jurkat T cells and human PBTs with Me 2 SO alone (Control) did not activate Erk1/2. It should be noted that we also examined the effects of 100 M (ϩ/Ϫ) Bay K 8644 on naïve human PBTs that were immediately isolated from PBMCs and not previously cultured with 10 g/ml plate-bound OKT3 and rhIL-2. Unstimulated naïve human PBTs had the same level of Erk2 activation following (ϩ/Ϫ) Bay K 8644 treatment as human PBTs grown for . The human PBTs contained 14% CD3 ϩ CD4 ϩ CD8 Ϫ , 79% CD3 ϩ CD4 Ϫ CD8 ϩ , 6.5% CD3 ϩ CD4 Ϫ CD8 Ϫ and 0.5% CD3 Ϫ CD4 Ϫ CD8 Ϫ cells. The results are representative of three independent experiments. 8 days in culture with OKT3 and rhIL-2 (data not shown).
Since there is very little information on what aspects of the T lymphocyte activation process are affected by inhibiting Ca 2ϩ influx with nifedipine treatment, we examined whether Erk1/2 activation induced by (ϩ/Ϫ) Bay K 8644 could be specifically blocked by nifedipine. Pretreatment of Jurkat T cells with either 100 M or 200 M nifedipine, but not the Me 2 SO solvent, significantly inhibited Erk1/2 activation induced by 100 M (ϩ/Ϫ) Bay K 8644 (Fig. 4C). As a positive control for Erk1/2 phosphorylation, we stimulated Jurkat T cells with soluble OKT3. Stimulation of Jurkat T cells through the TCR/CD3 complex induced robust activation of Erk1/2, showing that the Ca 2ϩ influx induced by (ϩ/Ϫ) Bay K 8644 supports only partial Erk1/2 activation.
Nifedipine Blocks NFAT-Transcriptional Activity in Anti-CD3-stimulated Jurkat T Cell Leukemia Line-The activation of the transcription factor NFAT is dependent upon increased intracellular Ca 2ϩ (36). We investigated whether the block in Ca 2ϩ influx through L-type Ca 2ϩ channels by nifedipine alters the transcriptional activity of NFAT by transiently transfecting an NFAT-luciferase reporter plasmid into Jurkat T cells. Activation of transfected Jurkat T cells induces endogenous NFAT transcription factors to bind to the NFAT cis-acting enhancer element within the construct, and transcribe the reporter gene. The maximum induction of NFAT when trans-fected Jurkat T cells were exposed to soluble OKT3 occurred between 5 to 8 h after stimulation (data not shown). Blocking Ca 2ϩ channel activity by pretreatment of Jurkat T cells with nifedipine resulted in inhibition of OKT3-induced NFAT activation in a dose-dependent manner (Fig. 5). Low concentrations of nifedipine that weakly blocked Ca 2ϩ influx, such as 50 M nifedipine, significantly reduced the transcriptional activity of NFAT. Higher doses of nifedipine, such as 200 M, almost completely abolished NFAT activity. The Me 2 SO solvent alone did not inhibit NFAT activation in Jurkat T cells. Additionally, OKT3 stimulated Jurkat T cells transiently transfected with a reporter construct that does not contain the NFAT cis-acting enhancer element (Control) did not activate NFAT.

IL-2 Production and IL-2R Expression is Inhibited by Nifedipine in Jurkat T Cell Leukemia Line and Human PBTs-
Since IL-2 secretion is a definitive indicator of T cell activation, we assessed whether blocking L-type Ca 2ϩ channels with nifedipine can inhibit IL-2 production in both anti-CD3 stimulated Jurkat T cells (Fig. 6A) and human PBTs (Fig. 6B). In agreement with the results showing the effect of nifedipine on Ca 2ϩ influx (Fig. 3) and NFAT activation (Fig. 5), nifedipine significantly inhibited IL-2 secretion in both cell types and abolished IL-2 secretion completely at 200 M nifedipine. The Me 2 SO solvent did not significantly block IL-2 secretion from either Jurkat T cells or human PBTs. To ensure the block in IL-2 secretion by nifedipine was not due to cell death induced by drug cytotoxicity, both Jurkat T cells and human PBTs were stained with PI after culture supernatants were removed for assaying IL-2. The PI negative or viable cell population was then analyzed by flow cytometry. In Jurkat T cells and human PBTs, 1-200 M nifedipine did not have a statistically significant impact on cell viability compared with the viability of cells stimulated with OKT3/TPA and treated with Me 2 SO (Fig. 6, C and D).
We then examined whether the inhibitory effect of nifedipine could be reversed by the addition of Ca 2ϩ . Since ionomycin rapidly increases intracellular Ca 2ϩ in T cells, treatment with this ionophore was used to provide additional Ca 2ϩ to the cells. Jurkat T cells (Fig. 6E) and human PBTs (Fig. 6F) were treated with nifedipine and ionomycin where indicated and IL-2 secretion was again assayed as an indicator of T cell activation. In both cell types, inhibition of IL-2 secretion by 1 M and 10 M nifedipine could be completely overcome by the addition of ionomycin. However, the treatment of T cells with 50 M nifedipine could only be partially reversed by ionomycin treatment. In all cases, reversing the inhibitory effect of nifedipine with ionomycin was more successful in human PBTs compared with Jurkat T cells.
IL-2R expression was also examined on viable T cells and it was found that only 200 M nifedipine significantly inhibited receptor expression. In Jurkat T cells (Fig. 7A), 200 M nife-dipine caused a 55% decrease in the log mean fluorescence intensity of IL-2R expression, whereas in human PBTs pretreated with 200 M nifedipine (Fig. 7B) a 70% decrease was observed compared with IL-2R expression of T cells stimulated with OKT3/TPA and treated with Me 2 SO.
Nifedipine Suppresses Splenocyte Proliferation-To investigate whether nifedipine could block the proliferation of T lymphocytes, we assayed the effects of nifedipine on the proliferative response induced by a mixed lymphocyte reaction (MLR). Nifedipine significantly suppressed the proliferation of splenocytes in a dose-dependent fashion (Fig. 8A). Low doses of nifedipine, including 1 and 10 M, weakly inhibited splenocyte proliferation, whereas 100 and 200 M nifedipine completely abrogated proliferation. There was no significant inhibition of splenocyte proliferation by treatment with Me 2 SO solvent alone.
Since nifedipine clearly inhibited MLR induced splenocyte proliferation in vitro, we evaluated whether nifedipine could block the proliferation of an antigen specific T cell response in vivo. To address this question we examined the proliferative response of female H-Y-specific TCR-Tg CD8 ϩ T cells transferred into male C57Bl/6 mice treated with either one dose of 15 mg/kg nifedipine or vehicle control. In male recipients, H-Yspecific CD8 ϩ T cells accumulating in the spleen proliferated in response to the male antigen. Treating male mice with one dose of 15 mg/kg nifedipine inhibited, but did not completely abro- The human PBTs contained 18% CD3 ϩ CD4 ϩ CD8 Ϫ , 69% CD3 ϩ CD4 Ϫ CD8 ϩ , 11.5% CD3 ϩ CD4 Ϫ CD8 Ϫ , and 1.5% CD3 Ϫ CD4 Ϫ CD8 Ϫ cells. Cell lysates were separated by SDS-PAGE and transferred to nitrocellulose. Membranes were probed with a phosphospecific Ab to detect activated Erk1/2 (top panel). The membrane was stripped and reprobed with an Ab directed against Erk1/2 to detect the total amount of kinase loaded in each lane (bottom panel). The results are representative of three independent experiments. gate, H-Y specific CD8 ϩ T cell proliferation (Fig. 8B). Nifedipine treatment resulted in an increased number of H-Y-specific CD8 ϩ T cells undergoing only 1-2 cell divisions and significantly fewer cells transiting to 3 divisions compared with the vehicle control. We addressed the specificity of nifedipine in this assay by determining whether administration of 15 mg/kg nifedipine 1 h (half-life of nifedipine in mice) prior to i.v. injection of the H-Y-specific thymocytes would lead to a difference in the T cell proliferation profile compared with vehicle control (37). In this experiment, H-Y-specific CD8 ϩ T cells proliferated to the same extent as the vehicle control, suggesting that nifedipine is specifically blocking L-type Ca 2ϩ channels and not inducing a nonspecific hormonal change in the mice, which could lead to decreased T cell proliferation (data not shown). We also assayed the proliferative response of H-Y-specific CD8 ϩ T cells in female mice as a control for no proliferation. As expected, the H-Y-specific CD8 ϩ T cells homed to the spleen but did not proliferate in the female mice due to the absence of the H-Y male antigen (Fig. 8B, Control, open bar). DISCUSSION The first step in the evaluation of the VDCC model was to provide molecular evidence that a functional L-type Ca 2ϩ channel protein is expressed in T lymphocytes. As the presence of mRNA frequently correlates with the expression of a protein, we have begun this investigation by demonstrating through nested RT-PCR that the L-type ␣ 1F -subunit gene, CACNA1F, is expressed in human T lymphocytes. Furthermore, the sequence of the PCR product unequivocally proves that the CACNA1F gene is expressed in T lymphocytes, which is a novel and exciting finding presently unreported in the literature.
In order to more directly assess the role of L-type Ca 2ϩ channels in T lymphocytes, we noted that previous studies have used synthetic DHP derivatives to study the function of these channels in the plasma membranes of numerous cell types (32,33,38). In the present study, we used the DHP derivatives, (ϩ/Ϫ) Bay K 8644 and nifedipine, to further investigate the presence of L-type Ca 2ϩ channels in T lymphocytes and to assess the contribution of Ca 2ϩ influx by L-type Ca 2ϩ channels during the T lymphocyte activation process. Through an intracellular Ca 2ϩ assay, we demonstrated that (ϩ/Ϫ) Bay K 8644 exerts an agonistic action on the Ca 2ϩ channels of Jurkat T cells and human PBTs. However, treatment with (ϩ/Ϫ) Bay K 8644 did not induce maximal Ca 2ϩ influx in either cell type since anti-CD3 stimulation of PBTs and Jurkat T cells resulted in a 2-4-fold larger increase in intracellular Ca 2ϩ , respectively. We also showed that nifedipine only partially blocks Ca 2ϩ influx, and does not significantly effect Ca 2ϩ release from intracellular Ca 2ϩ stores in Jurkat T cells following stimulation through the TCR/CD3 complex. Therefore we conclude that nifedipine is blocking L-type Ca 2ϩ channels found in the plasma membrane, with minor inhibitory effects at higher concentrations on Ca 2ϩ release from intracellular stores.
Although the specificity of action of DHPs has been confirmed by determining the DHP binding sites on the channelforming ␣ 1 -subunit of L-type VDCCs (39,40), there are studies demonstrating the nonspecific inhibitory effects of high micromolar concentrations of DHPs on voltage-dependent potassium (K v ) channels and Ca 2ϩ -activated potassium (K ϩ ) channels (41)(42)(43). Even though these observations could be of concern in the present study, Fagni et al. (42) reported that micromolar concentrations of both nifedipine and the stereospecific enantiomers of Bay K 8644 exhibited an inhibitory effect on K ϩ current through Kv and Ca 2ϩ -activated K ϩ channels. This is in contradiction to our results, since we show that (ϩ/Ϫ) Bay K 8644 has an agonistic effect while nifedipine antagonizes Ca 2ϩ influx in T lymphocytes. In addition (ϩ/Ϫ) Bay K 8644 does not activate K ϩ channel currents (42). Therefore, we conclude that the overall observed effect of the DHPs in this study was due to modulation of Ca 2ϩ influx through L-type Ca 2ϩ channels. Minor inhibitory effects of DHPs on K ϩ current may occur at the higher micromolar concentrations, but are reportedly absent at the lower range of nifedipine used in this study where inhibition of Ca 2ϩ influx is still observed (41).
After confirming that (ϩ/Ϫ) Bay K 8644 and nifedipine can mediate Ca 2ϩ influx in T lymphocytes, we investigated whether Ca 2ϩ influx through an L-type Ca 2ϩ channel can modulate early Ca 2ϩ -dependent signaling events, such as MAP kinase activity. It has been postulated that Ca 2ϩ can interact with the MAP kinase signaling pathway in T lymphocytes by activating Lck and calmodulin-kinase, which are upstream of Erk1/2 and responsible for Ca 2ϩ -dependent Erk1/2 enzymatic activation (44). We report here that (ϩ/Ϫ) Bay K 8644 could induce phosphorylation and activation of both Erk1 and Erk2 in Jurkat T cells, weak activation of Erk2 in human PBTs, and that the activation of Erk1/2 by (ϩ/Ϫ) Bay K 8644 could be blocked by pretreatment with nifedipine. These results support the hypothesis that Ca 2ϩ influx through an L-type Ca 2ϩ channel mediates the MAP kinase signaling pathway during T lymphocyte activation. The quantitative discrepancy between Erk activation in Jurkat T cells and primary T lymphocytes might occur if the human PBTs required a higher sustained level of intracellular Ca 2ϩ to induce Erk activity compared with transformed T lymphocytes. We observed that the Ca 2ϩ ionophore, ionomycin, induces a 2.5-fold greater increase in intracellular Ca 2ϩ in human PBTs compared with Jurkat T cells, which corresponds with a more robust phosphorylation of Erk1/2 by ionomycin in human PBTs. In addition, Ca 2ϩ entry induced by (ϩ/Ϫ) Bay K 8644 in human PBTs was only transient compared with the sustained Ca 2ϩ influx in Jurkat T cells. Therefore the difference in the amount of Ca 2ϩ influx could result in (ϩ/Ϫ) Bay K 8644 strongly activating Erk1/2 in The human PBTs in this experiment contained 11.5% CD3 ϩ CD4 ϩ CD8 Ϫ , 86.5% CD3 ϩ CD4 Ϫ CD8 ϩ , 1.0% CD3 ϩ CD4 Ϫ CD8 Ϫ , and 1.0% CD3 Ϫ CD4 Ϫ CD8 Ϫ cells. *, p Ͻ 0.01, as comparing samples with or without ionomycin added. Results depicted are representative of three independent experiments. Each bar represents the mean and S.D. of assays from triplicate wells. expression. We demonstrated that inhibiting Ca 2ϩ influx with nifedipine could inhibit the activity of NFAT in a dose-dependent manner in Jurkat T cells. Since NFAT regulates the transcription of several cytokine genes, including IL-2, we then examined the effects of nifedipine on IL-2 production and IL-2R expression (36). In both Jurkat T cells and human PBTs, IL-2 production was blocked in the presence of nifedipine. We confirmed that the overall inhibition in IL-2 secretion mediated by nifedipine was due to a block in Ca 2ϩ influx and not cell death since the percentage of viable cells did not significantly change with increasing nifedipine dose. These results are consistent with a previous report showing that inhibition of human T lymphocyte proliferation by 100 M nifedipine was not due to drug cytotoxicity (23). We also demonstrated that the block in IL-2 secretion by nifedipine can be reversed by the addition of ionomycin, confirming that low doses of nifedipine are inhibiting L-type Ca 2ϩ channels and not suppressing the function of other channels in T lymphocytes. IL-2R expression was only downregulated with high concentrations of nifedipine. This is consistent with the current understanding that the signaling requirements for expression of the IL-2R are less stringent than those for IL-2 production (45). Thus Ca 2ϩ entry through L-type Ca 2ϩ channels regulates NFAT activity and IL-2 production but has a lesser effect on IL-2R expression.
In addition to demonstrating that an L-type Ca 2ϩ channel mediates Ca 2ϩ influx during T lymphocyte activation, we also investigated whether an L-type Ca 2ϩ channel would regulate the proliferation of T lymphocytes. To this end, we assayed the proliferation of mouse splenocytes through an in vitro MLR and found that nifedipine markedly inhibited mouse splenocyte proliferation in a dose-dependent manner. Although the in vitro anti-proliferative effects of nifedipine have been documented, there are no previous studies describing nifedipinemediated inhibition of T cell proliferation in vivo. We are the first to report that one dose of 15 mg/kg nifedipine treatment specifically slows down, but does not completely abolish the proliferation of H-Y-specific TCR-Tg CD8 ϩ T lymphocytes in mice. These observations support our hypothesis that Ca 2ϩ influx through L-type Ca 2ϩ channels is required for sustained Ca 2ϩ influx during T lymphocyte proliferation in vitro and in vivo.  Taken together, the results in this study show that (ϩ/Ϫ) Bay K 8644 and nifedipine partially modulate T lymphocyte activation and proliferation. Although we have provided evidence for the presence of a DHP sensitive L-type Ca 2ϩ channel in the plasma membrane of T lymphocytes, additional Ca 2ϩ channels may also contribute to the Ca 2ϩ influx pathway. This conclusion is in agreement with recent studies, describing the involvement of TRP ion channels in regulating Ca 2ϩ influx in T lymphocytes. Cui et al. (15) showed that the TRP-vanilloid receptor family member of ion channels, CaT1, is involved in generating I CRAC in Jurkat T cells, which is partially regulated through intracellular Ca 2ϩ store depletion. Although CaT1 plays a significant role in mediating Ca 2ϩ entry, overexpression of a dominant negative pore-region mutant of CaT1 did not completely abolish Ca 2ϩ influx in Jurkat T cells, leading to the possibility that other channels, such as L-type Ca 2ϩ channels are also involved in Ca 2ϩ entry (15). TRP channels that are not modulated by intracellular Ca 2ϩ store depletion have also been found in human T lymphocytes. For instance, Sano et al. (16) reported that the LTRPC2 protein is abundantly expressed in human peripheral blood and Jurkat T cells and mediates Ca 2ϩ influx in response to elevated levels of pyrimidine nucleotides, adenosine 5Ј-diphosphoribose, and NAD. TRP6 mRNA and protein is also expressed in Jurkat T cells and PBTs and Ca 2ϩ influx through this channel is activated by diacylglycerol (14). In conjunction with the results from this study and the recent discovery of TRP protein expression in T lymphocytes, it is highly probable Ca 2ϩ entrance into T lymphocytes is mediated through multiple Ca 2ϩ channels, including both TRP and Ltype Ca 2ϩ channels. Given that the amplitude and duration of Ca 2ϩ signals in T lymphocytes are very diverse, a number of different channels may be necessary to coordinate the different Ca 2ϩ responses required for T lymphocyte activation, proliferation and death.
Our study also raises the question of whether administration of DHPs, such as nifedipine, for treatment of cardiovascular disorders has deleterious side effects on circulating T lymphocytes (46). Currently nifedipine is widely prescribed by clinicians in the treatment of cardiovascular disorders, including hypertension and ischemic stroke (38,47). In patients with cardiovascular disease, nifedipine blocks the action of L-type VDCCs, which are responsible for initiating the contraction of cardiac and smooth muscles (38). Interestingly, a single study has reported that lymphocytes in healthy humans treated with one 10 mg oral dose of nifedipine have decreased blastogenesis and IL-2 production (48). We have shown that one 15 mg/kg dose of nifedipine can suppress T lymphocyte proliferation in mice. Even though the doses of nifedipine in the present study are higher than the serum concentration of patients administered DHPs (49), long term treatment with nifedipine may act as an immunosuppressant, affecting the immune competence of patients with cardiovascular disorders. Our present findings should encourage further research into determining whether cardiac patients receiving Ca 2ϩ channel blockers are immunosuppressed.
In summary, we have shown both molecular and extensive pharmacological evidence for the presence of an L-type Ca 2ϩ channel in T lymphocytes. Elucidation of the role of the L-type Ca 2ϩ channel ␣ 1F -subunit will provide the basis for a better understanding of the mechanisms controlling Ca 2ϩ influx in T lymphocytes and may lead to the development of novel therapeutic agents to control T lymphocyte activation and inactivation states. The results from the present study clearly support the hypothesis that an L-type Ca 2ϩ channel is present in the plasma membrane of T lymphocytes and that this channel contributes to Ca 2ϩ influx during T lymphocyte activation and proliferation. FIG. 8. Nifedipine suppresses splenocyte proliferation. A, splenocytes from C57Bl/6 mice were incubated with 1-200 M nifedipine in triplicate and then stimulated with irradiated Balb/c splenocytes. 5-6 days later, each sample was assayed for proliferation as reflected as lymphocyte number determined by flow cytometry. The control is splenocytes treated with Me 2 SO (DMSO) solvent alone. Each bar represents the mean and S.D. of assays from triplicate wells. p Ͻ 0.01, relative to the Control. B, CFSE-loaded thymocytes from C57Bl/6 female mice with Tg TCR␣␤ H-Y receptor were i.v. injected into female (Control, n ϭ 1) or male recipients. After 20 -24 h, male mice received one intraperitoneal dose of vehicle (n ϭ 4) or 15 mg/kg nifedipine (n ϭ 4). Splenocytes were harvested 40 h after the initial i.v. injection and proliferation of CFSE ϩ , CD8 ϩ , and Tg TCR high splenocytes was quantified by flow cytometry. Bars represent percent of total cells exhibiting a discrete CFSE (FL1) intensity reflecting an equal number of cell divisions: no divisions (open bar), 1 division (solid bar), 2 divisions (hatched bar), and 3 divisions (dotted bar). Each bar represents the mean and S.D. of assays from four mice. *, p Ͻ 0.01, as comparing cell divisions between vehicle control and nifedipine treated male mice. The results depicted are representative of three independent experiments.