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T Cells Express 7-Nicotinic Acetylcholine Receptor Subunits That Require a Functional TCR and Leukocyte-Specific Protein Tyrosine Kinase for Nicotine-Induced Ca²⁺ Response¹

Seddigheh Razani-Boroujerdi^*, R. Thomas Boyd, Martha I. Dávila-García, Jayashree S. Nandi^*, Neerad C. Mishra^*, Shashi P. Singh^*, Juan Carlos Pena-Philippides^*, Raymond Langley^* and Mohan L. Sopori^2,*

^* Immunology Division, Lovelace Respiratory Research Institute, Albuquerque, NM 87108; Department of Neuroscience, College of Medicine and Public Health, The Ohio State University, Columbus, OH 43210; and Department of Pharmacology, College of Medicine, Howard University, Washington, DC 20059

	Abstract

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Acute and chronic effects of nicotine on the immune system are usually opposite; acute treatment stimulates while chronic nicotine suppresses immune and inflammatory responses. Nicotine acutely raises intracellular calcium ([Ca²⁺]_i) in T cells, but the mechanism of this response is unclear. Nicotinic acetylcholine receptors (nAChRs) are present on neuronal and non-neuronal cells, but while in neurons, nAChRs are cation channels that participate in neurotransmission; their structure and function in nonexcitable cells are not well-defined. In this communication, we present evidence that T cells express 7-nAChRs that are critical in increasing [Ca²⁺]_i in response to nicotine. Cloning and sequencing of the receptor from human T cells showed a full-length transcript essentially identical to the neuronal 7-nAChR subunit (>99.6% homology). These receptors are up-regulated and tyrosine phosphorylated by treatment with nicotine, anti-TCR Abs, or Con A. Furthermore, knockdown of the 7-nAChR subunit mRNA by RNA interference reduced the nicotine-induced Ca²⁺ response, but unlike the neuronal receptor, -bungarotoxin and methyllycaconitine not only failed to block, but also actually raised [Ca²⁺]_i in T cells. The nicotine-induced release of Ca²⁺ from intracellular stores in T cells did not require extracellular Ca²⁺, but, similar to the TCR-mediated Ca²⁺ response, required activation of protein tyrosine kinases, a functional TCR/CD3 complex, and leukocyte-specific tyrosine kinase. Moreover, CD3 and 7-nAChR coimmunoprecipitated with anti-CD3 or anti-7-nAChR Abs. These results suggest that in T cells, 7-nAChR, despite its close sequence homology with neuronal 7-nAChR, fails to form a ligand-gated Ca²⁺ channel, and that the nicotine-induced rise in [Ca²⁺]_i in T cells requires functional TCR/CD3 and leukocyte-specific tyrosine kinase.

	Introduction

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The nicotinic acetylcholine receptors (nAChRs)³ are ligand-regulated, ion-channel complexes that mediate neurotransmission, presynaptic control of neurotransmitters, and second messenger cascades in the central and peripheral nervous system (1, 2, 3). Functional nAChRs are believed to be pentameric complexes. In vertebrates, 12 neuronal nAChR subunits have been identified (2–10 and β2–4). Most nAChRs are composed of multimeric complexes; however, 7, 8, and 9 nAChR subunits form functional homopentameric receptors (4, 5, 6). On neurons, the ion channel formed by 7-nAChRs is assembled from five homomeric subunits and permeates primarily Ca²⁺ (7, 8). 7-nAChRs have also been found in invertebrates such as Caenorhabditis elegans and Drosophila (9, 10, 11). In mammals, these receptors are also widely expressed in nonexcitable cells such as keratinocytes (12), lung type-2 epithelial cells (13, 14), and macrophages (15), but their structure and function on non-neuronal cells are not clearly defined. Although in neurons, the 7-nAChRs participate in neurotransmission which is blocked by -bungarotoxin (-BTX) and methyllycaconitine (MLA) (16, 17), it is unlikely that these receptors perform such a function in nonexcitable cells.

Chronic in vivo nicotine treatment suppresses the immune and inflammatory responses (18, 19, 20), causing T cell anergy (21) and decreased leukocyte migration; however, acute exposure to nicotine increases intracellular calcium [Ca²⁺]_i and cell migration in several cell types (18, 14, 22, 23). In vivo, nicotine regulates T cell function by direct interaction with T cells and indirectly through neuroimmune interactions (18, 24). RT-PCR analysis suggests the expression of 7-like nAChR nucleotide sequences in T cells (25, 26, 27, 28, 29), but their structure and function have not been clearly identified. Exposure of T cells to nicotine stimulates protein tyrosine kinase (PTK) activities and raises the [Ca²⁺]_i concentration (18), indicating that T cells respond directly to nicotine. It is generally assumed that nAChRs on all cell types, including those on non-neuronal cells, are cation channels (28, 30), but no electrophysiological evidence supports the presence of nicotine-sensitive, ligand-gated cation channels on T cells. Recent evidence suggests that 7-nAChRs on some neuronal cells fail to raise nicotine-induced [Ca²⁺]_i (31), and in astrocytes the nicotine-induced rise in [Ca²⁺]_i is amplified by Ca²⁺-induced Ca²⁺ influx (32, 33). TsA201 cells transfected with 7-nAChR cDNA express 7-nAChRs on the cell surface, but do not produce nicotine-induced inward currents (34). Moreover, in mammalian cells 7-nAChRs might require additional proteins to form a functional cationic channel (35, 36). Thus, the mere presence of 7-nAChR transcripts or membrane-localized 7-nAChR subunits is not sufficient to generate a functional nicotine-sensitive cation channel. In this study, we present evidence that although T cells express a full-length 7-nAChR subunit transcript that is essentially identical in the nucleotide sequence to the human neuronal 7-nAChR subunit, it does not result in the formation of a functional ligand-gated ion channel. Moreover, the nicotine-induced rise in the [Ca²⁺]_i, thought to be mediated by this 7-nAChR, requires a functional TCR/CD3 complex and the Src PTK, leukocyte-specific tyrosine kinase (Lck).

	Materials and Methods

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Cell lines

Wild-type (WT) Jurkat cells, Lck-deficient (Lck^–) Jurkat cells, CD3-β-mutant (β^–) Jurkat cells, and the rat pheochromocytoma cell line PC12 were purchased from American Type Culture Collection and maintained in complete medium (RPMI 1640 containing 10% FCS, 50 mM 2-ME, 2 mM glutamine, and 10 µg/ml gentamicin) at 37°C in 5% CO₂.

Isolation of rat splenic T cells

Rat spleen cells were prepared essentially as described elsewhere (21). Briefly, spleens were passed through stainless steel mesh and the RBC were lysed with NH₄Cl solution. After washing, the pellet was resuspended in MACS buffer (PBS (pH 7.2) containing 2 mM EDTA and 0.5% FBS). The subsequent T cell purification steps were conducted in the cold (4–6°C). Briefly, spleen cells were incubated with rat pan T Cell MACS beads (Miltenyi Biotec) for 15 min. Beads were washed and resuspended in MACS buffer, and then loaded onto a column for magnetic separation of T cell-containing beads from unbound cells. Cells retained on the beads were washed twice with cold MACS buffer and then suspended in an appropriate buffer. The purified cells were >98% CD3⁺ as analyzed by flow cytometry.

Isolation of human T cells (hT)

The Lovelace Respiratory Research Institute Institutional Review Board approved the use of human blood from normal adult volunteers. PBMCs were obtained from heparinized blood by standard density gradient centrifugation on Histopaque (Sigma-Aldrich); after washing, the cell pellet was suspended in MACS buffer (PBS (pH 7.2) containing 2 mM EDTA and 0.5% FBS). Purified hT cells were isolated from the PBMCs by incubating with human anti-CD3 MACS beads (Miltenyi Biotec) as described above for the purification of rat T cells. The cells were >98% CD3⁺ as analyzed by flow cytometry.

RT-PCR for 7-nAChR expression in Jurkat cells

RT-PCR for 7-nAChRs was conducted essentially as described previously (14). Briefly, Jurkat cells were homogenized in Tri reagent. (MRC Molecular Research Center, Cincinnati, OH). Total RNA was isolated using the BCP phase separation reagent (MRC Molecular Research Center). RNA was precipitated by 2-propanol and washed with 75% ethanol. The RNA pellet was dried for 30 min, resuspended in RNase-free water, and quantitated spectrophotometrically. The gene-specific primer sets for 7-nAChRs and GAPDH were purchased from Sigma-Genosys (www.sigma-genosys.com). The primer set for human 7-nAChRs was as follows: sense primer (5'-CCCGGCAAGAGGAGTGAAAGGT-3') and antisense primer (5'-TGCAGATGATGGTGAAGACC-3'), generating an 843-bp 7 band. The primer set for human GAPDH was as follows: upstream primer (5'-ACACATGCCATCACTGCC-3') and downstream primer (5'-GCCTGCTTCACCACCTTCTTG-3'), resulting in a PCR product of 266 bp. RT-PCR was conducted with a Qiagen RT-PCR kit, according to the manufacturer’s directions. Briefly, cDNA synthesis and predenaturation were performed by one cycle of 50°C for 30 min and 94°C for 1 min. The cDNA was amplified by 35 PCR cycles (denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min), and the final extension of one cycle of 72°C for 10 min in a Thermal Cycler 9600 (PerkinElmer). The PCR products were electrophoresed on 3% agarose gel with ethidium bromide staining for visualization. The gel was photographed; the bands were quantified with a Bio-Rad GS-800 scanner and Quantity One software and standardized to the housekeeping gene GAPDH to present the level of gene expression.

Cloning and sequencing of 7-nAChR from T cells

Tri reagent (Medical Research Center, Cincinnati, OH) was used to extract RNA from hT; WT, Lck^–, and β^– Jurkat cells; and purified rat T cells. Jurkat cell poly(A) RNA was also purchased from Ambion. Primers were designed from the neuronal 7-nAChR subunit sequences from the GenBank database (NM_000746) and purchased from Invitrogen Life Technologies.

For the RT step, a gene-specific downstream (reverse) primer was used. After the initial denaturation at 65°C for 5 min, RNA, dNTPs, and the gene-specific downstream (reverse) primer were incubated at 60°C for 60 min. Thermoscript RT-PCR or Superscript RT systems were used; for the PCR step, Hifi Taq polymerase (Invitrogen Life Technologies) with forward and reverse primers in different combinations yielded PCR products of various sizes. Table I presents the location and sequences of primers for translated and untranslated regions used to amplify 7 transcripts from Jurkat and hT cells. The PCR products were analyzed by agarose gel electrophoresis and appropriate product sizes were purified by gel extraction (Qiagen). The purified PCR products were cloned in pCRII-TOPO vectors (Invitrogen Life Technologies) according to the manufacturer’s instructions. "One shot" competent Escherichia coli cells were transformed and recombinant clones were selected. Miniprep DNAs from the purified plasmids (Qiagen) were digested with EcoRI to release the insert to confirm the right size of the inserts. The cloned DNA samples were sequenced using vector-specific (M13) forward and reverse primers on both strands and the sequences were submitted to GenBank (Jurkat original: accession number: AY909581; Lck^– Jurkat, accession number: AY909582. Other PCR product sequences from various axons are in the process of being submitted to GenBank).

View this table: [in this window] [in a new window]   Table I. Sequence of primers from various regions of 7 subunit

Nicotine and Con A treatment of rat T cells and Jurkat cells

Cells (5 x 10⁵ cells/ml) were cultured in the presence or absence of Con A (5 µg/ml) or nicotine (10 µM). The cultures were incubated at 37°C for 24 h in a 5% CO₂ atmosphere. Cells were harvested and stained with 100 nM Alexa Fluor 488-conjugated -BTX (Molecular Probes) for 1 h. After washing with cold PBS, cells were analyzed by flow cytometry.

Jurkat cell membrane preparations and epibatidine binding

The membrane homogenates were prepared from normal and mutant Jurkat cells as previously described (37). Briefly, cells were collected and homogenized in 50 mM Tris buffer (pH 7.0) at 4°C. The protein content of the cell suspension was determined by the BCA method (Pierce Biotechnology). Protein (50–100 µg) was added to tubes containing 50 µl of 300 pM [³H]epibatidine ([³H]EB), a nonselective nicotinic receptor agonist ligand. Nonspecific binding was measured in tubes containing 300 µM nicotine hydrogen tartrate, and the specific binding was calculated as the difference between total and nonspecific binding.

Determination of PTK, Lck, and Fyn kinase activities in Jurkat cells in response to nicotine

PTK activity of cell lysates was determined essentially as described elsewhere (21). Briefly, Jurkat cells were incubated with 1 µM nicotine in complete medium at 37°C for the stated times. Where indicated, cells were preincubated with 20 µM genistein for 1 h before nicotine treatment. Cells were lysed with ice-cold lysis buffer (50 mM Tris-HCl (pH 7.2); 150 mM NaCl; 1 mM sodium orthovanadate; 1 mM PMSF; 10 µg/ml aprotinin; 10 µg/ml leupeptin; and 10 mM NaF) containing 1% Brij-96 for 20 min, and the lysates were clarified by centrifugation. The protein concentration of lysates was determined by the BCA protein assay kit (Pierce). For total PTK activity, 20 µl of lysates equivalent to 10–20 µg of protein were boiled in Laemmli sample buffer and electrophoresed on 12.5% SDS-PAGE. Proteins were transferred on a nitrocellulose paper, the blots were stained with Ponceau S to confirm equal loading of the receptor in each well, and then blots were blocked with 3% BSA in TBST (20 mM Tris, 500 mM NaCl, 0.05% Tween 20 (pH 7.5)), and probed with anti-phosphotyrosine Ab. Where indicated, the membranes were stripped using the Restore Western Blot Stripping Buffer (Pierce). Briefly, blots were washed to remove chemiluminescent substrate and incubated in the stripping buffer for 30 min at room temperature. The membranes were removed, washed in TBS, blocked with 3% BSA in TBST, and probed with anti-actin Ab (Santa Cruz Biotechnology) as described earlier. For Fyn and Lck activities, lysate aliquots equivalent to 150 µg of protein were incubated for 2 h at 4°C with either 5 µl of anti-Fyn or 2 µg/ml anti-Lck Abs (Santa Cruz Biotechnology). Immune complexes were collected by adding 30 µl of 50% protein A Sepharose for 30 min at 4°C, followed by four washes with wash buffer. Fyn and Lck immune complexes were washed with Fyn kinase reaction buffer (30 mM HEPES (pH 7.2); 3.5 mM MnCl₂; and 3.7 mM MgCl₂) and Lck kinase reaction buffer (5 mM HEPES (pH 7.4); 5 mM p-nitrophenyl phosphate; and 10 mM MnCl₂), respectively. The kinase activity of Fyn and Lck immune complexes was determined in a total of 25 µl of the respective kinase reaction buffers. Although the autophosphorylation reaction of Lck was initiated by the addition of 10 µCi [-³²P]ATP, Fyn kinase activity was started with 10 µCi [-³²P]ATP and 2 µg of acid-denatured enolase. After incubation at 30°C for 10 min, reactions were stopped by heating and the components were resolved by 10% SDS-PAGE. Gels were dried and the phosphoproteins were visualized by autoradiography.

Coimmunoprecipitation and Western blot analysis of 7-nAChRs

Cells were lysed in ice cold lysis buffer (3 mM Na₃VO4; 15 mM Na₄P₂O₇; 150 mM NaF; 6 mM iodoacetate; 15 mM EDTA; 0.3% TX-100; 150 mM NaCl; 150 mM Tris-HCl (pH 7.4); 3 mM PMSF; and 30 µM each of aprotinin, leupeptin, and pepstatin). Cell lysates were precleared by adding appropriate control IgG and agarose-conjugated protein A beads, and the suspensions were centrifuged. The supernatants were immunoprecipitated with a 7-nAChR-specific polyclonal Ab, CD3-- specific mAb, or the isotype control Igs (Santa Cruz Biotechnology). The immunoprecipitates were captured by protein A beads, eluted by boiling, and run on 12.5% Tris-HCl Criterion Precast Gels (Bio-Rad) along with Kaleidoscope Prestained Standards (Bio-Rad). The gels were blotted, blocked with dry milk, and probed either with HRP-conjugated CD3--specific mAb (Santa Cruz Biotechnology) or with anti-7-nAChR subunit-specific Ab followed by HRP-labeled second Ab (Abcam). Blots were photographed and quantitated by a Fluor-S MultiImager system.

Assay for [Ca²⁺]_i

[Ca²⁺]_i was determined by previously described procedures (38). Briefly, cells (5 x 10⁶) were loaded with indo-1-AM, or indo-1-AM together with BAPTA-AM (Molecular Probes) for 20 min. After washing, cells were resuspended in Ca²⁺-containing medium (10 mM HEPES (pH 7.4); 126 mM NaCl; 3 mM KCl; 1.25 mM NaH₂PO₄; 26 mM NaHCO₃; 10 mM dextrose; 2 mM Ca²⁺; 1 mM Mg²⁺; and 5% FBS, adjusted to an osmolarity of 285) or Ca²⁺-free medium (same medium without Ca²⁺ with 5 mM EGTA). The [Ca²⁺]_i was determined by spectrofluorometry (Deltascan Model 4000; PTI). Because various cell types show different baseline [Ca²⁺]_i, it was recorded before the addition of test compounds or Abs and subtracted from test results. Changes in [Ca²⁺]_i were calculated as described elsewhere (38).

Knockdown of 7-nAChRs in Jurkat cells by small interfering RNA (siRNA) and antisense RNA

The siRNAs targeting human 7-nAChRs were purchased from Qiagen. The sequences were: sense (GUAGGAAAUGCGCAGAUAA)-dTdT and antisense (UUAUCUGCGCAUUUCCUAC)-dTdT. The antisense RNA (GCA GCG CAT GTT GAG TCC CG) targeting human 7-nAChR was purchased from Sigma-Genosys. The oligonucleotides were electroporated into cells using an Amaxa Nucleofector Device and the Nucleofector kit from Amaxa Biosystems. At 36 h postnucleofection, the cells were examined for 7-nAChR expression by RT-PCR, -BTX binding, and Western blot analysis.

	Results

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T cell membranes bind [³H]EB

Epibatidine is a nonselective ligand for nAChRs, including various (2, 3, and 4) subunits in combination with β2 or β4 (39) and the 7-homopentameric receptor (40). To determine whether nAChRs are expressed on T cells, cell membrane homogenates from normal Jurkat cells and the two mutated Jurkat cell lines, Lck^– and β^–, were incubated with 2 nM [³H]EB. Nonspecific binding was determined in the presence of a large excess of unlabeled nicotine (100 µM) and subtracted from the total binding to calculate [³H]EB-specific binding (37). Fig. 1 shows specific [³H]EB binding on all three Jurkat cell lines, suggesting the presence of nicotinic-binding sites on T cell membranes. Specific [³H]EB binding was also seen in membrane preparations from normal rat splenic T cells (data not shown).

View larger version (11K): [in this window] [in a new window]   FIGURE 1. Epibatidine binds normal and mutant (Lck– and β–) Jurkat cell lines. Jurkat cell membrane homogenates were prepared and treated with [3H]EB as described in Materials and Methods. All measurements were done in triplicate, and the graph represents six to seven independent experiments.

T cells have 7-nAChRs that are tyrosine phosphorylated by nicotine

To determine whether 7-nAChRs are present on T cells, rat spleen cells were analyzed for binding of the 7-nAChR-specific antagonist -BTX using Alexa Fluor 488-conjugated -BTX, and the cells were analyzed by flow cytometry (FACS). Fig. 2A (left panel) shows that a significant fraction of normal splenic cells bound -BTX, and the proportion of splenic cells (percentages given in the figure) that bound -BTX increased after overnight incubation with Con A (Fig. 2A, middle panel) and nicotine (Fig. 2A, right panel). Essentially, 100% of Jurkat cells bound -BTX (data not shown). Western blots of cell extracts isolated from rat spleen cells incubated overnight with either medium alone (CON) or 1 µM nicotine (NT) showed increased expression of 7-nAChR protein after nicotine treatment (Fig. 2B). Western blots of cell extracts from normal human peripheral blood T cells (hT cells), Jurkat cells, and rat brain homogenates, when probed with anti-7-nAChR mAb, showed the presence of 58-kDa protein in these cells (Fig. 2C). A similar 58-kDa protein was present in purified rat splenic T cell extracts, and the protein exhibited tyrosine phosphorylation when probed with anti-phosphotyrosine Abs (Fig. 2D). Tyrosine phosphorylation of the protein increased significantly if control T cells (Fig. 2D, right lane) were incubated for a short time (60 min) with either 1 µM nicotine (Fig. 2D, middle lane) or 5 µg/ml anti-TCR Ab (Fig. 2D, left lane). These results suggest that human and rat T cells contain a 58-kDa 7-nAChR immunoreactive protein that is tyrosine phosphorylated after treatment of T cells with anti-TCR Ab or nicotine.

View larger version (41K): [in this window] [in a new window]   FIGURE 2. Characteristics of nAChRs on T cells. A, Rat spleen cells were cultured for 24 h without (CON) or with Con A (5 µg/ml) or 10 µM nicotine (NT). After washing, cells were labeled with Alexa Fluor 488-conjugated -BTX and analyzed by flow cytometry as described in Materials and Methods. The percentage of -BTX bound cells is shown in each panel. B, Spleen cell extracts from cells incubated overnight with PBS (CON) or with 1 µM NT were analyzed by Western blots probed with anti-7-nAChR Ab. C, Cell extracts (10 µg of protein) from rat brain, WT Jurkat, and hT cells (three healthy human volunteers) were analyzed by Western blot analysis using 7-nAChR mAb. Jurkat cell extracts were also probed with isotype control Ig (isotype). D, Purified rat splenic T cells were incubated with 1 µM NT or anti-β-TCR Abs for 10 min; the cell extracts were resolved on SDS-PAGE and probed with anti-phosphotyrosine Abs as described in C. Except for hT cells (C), experiments presented in this figure were repeated at least three times.

Human T cells contain 7-nAChRs with high sequence homology to neuronal 7-nAChR

The human neuronal 7-nAChR gene is encoded by 10 exons, and the sequence analysis of the PCR products indicates that RNA similar to the human neuronal 7-nAChR is expressed in hT cells, human T cell lines (Jurkat cells), and in rat T cells (data not shown). RNA preparations from hT cells and Jurkat cells amplified with primer pairs from exons 2 and 7 generated a product that matched the human neuronal 7-nAChR sequence. A nested PCR using primers from 5' untranslated and 3' untranslated regions followed by amplification with a primer pair from exons 2 and 9 also detected a neuronal type 7-nAChR subunit cDNA in human T cells. This indicates that a full-length 7-nAChR RNA is expressed in hT cells and human T cell lines. In addition to the full-length product, RNA from WT Jurkat cells amplified by primer pairs from exons 7 and 10 generated two shorter PCR products that matched the human neuronal 7-nAChR subunit cDNA, but lacked either exon 9 or exons 8 and 9. Sequence analysis suggested that the alternatively spliced variants had the correct reading frame but terminated prematurely. However, the amino acid sequence of these "alternate" transcripts did not match any sequence in the GenBank, and similar spliced products were not seen in the RNA from another WT Jurkat cell clone. Thus, while T cells contain normal 7-nAChR subunit transcripts, some T cell lines may generate small amounts of alternatively spliced transcripts that are unlikely to make functional proteins.

The sequence similarity and divergence of the 7-nAChR subunit from hT cells, WT Jurkat cells, and Jurkat cell variants (Lck^–, β^–) were compared with the neuronal cells. Phylogenetic analyses (Fig. 3) indicated a very high homology (99.6%) between 7-nAChR subunits from hT, neuronal cells, and various Jurkat cell types, including Lck^– and β^– clones. Thus, the primary structure of the 7-nAChR subunit on neuronal cells and T cells is almost identical. Moreover, detailed nucleotide sequence alignment of the 7-nAChR subunit sequence from Jurkat cell lines using the ClustalW program and pairwise sequence alignment with the neuronal 7-nAChR subunit sequence showed some point mutations, but they were unlikely to affect the predicted amino acid sequences (data not shown). Minor sequence variations and genetic polymorphism in 7-nAChR transcripts are normally found in neurons (41, 42).

View larger version (19K): [in this window] [in a new window]   FIGURE 3. Nucleotide sequence homology between neuronal and T cell 7-nAChRs. ClustalW and DNASTAR programs were used to compare nucleotide sequences of 7-nAChRs between the indicated human T cells/T cell lines and compared with the human neuronal 7-nAChR sequence from GenBank as the reference. Comparison of the nucleotide sequence to self (100% identity with 0% divergence) is indicated as the filled box. The neuronal sequence matched the sequence of RT-PCR products from various T cells (row 1): hT (99.7%), WT (99.9%), Lck– (99.7%), and β– (99.8%) with divergence (column 1) of 0.3, 0.1, 0.0, and 0.2%, respectively. Similarly, β– Jurkat cells differed from Lck–, WT, and hT cells by 0.2, 0.3, and 0.1%, respectively (bottom row).

Nicotine increases PTK-dependent [Ca²⁺]_i in T cells that is insensitive to -BTX and MLA

We have previously shown that nicotine increases PTK activity and [Ca²⁺]_i in purified rat splenic T cell cultures (18). To investigate the relationship between nicotine-induced activation of PTK activity and rise in [Ca²⁺]_i in T cells, we compared the effects of the PTK inhibitor genistein on the nicotine-induced [Ca²⁺]_i response in Jurkat and PC12 cells. Although preincubation of Jurkat cells with 20 µM genistein for 1 h significantly reduced the nicotine-induced rise in [Ca²⁺]_i (Fig. 4A), genistein did not detectably affect the [Ca²⁺]_i response in PC12 cells (Fig. 4B). Similarly, genistein inhibited the nicotine-induced [Ca²⁺]_i response of purified rat splenic T cells (data not shown). Therefore, unlike the response in PC12 cells, the nicotine-induced [Ca²⁺]_i response in Jurkat cells is dependent on the activation of PTK activity.

View larger version (32K): [in this window] [in a new window]   FIGURE 4. Genistein blocks nicotine-induced rise in [Ca2+]i in Jurkat but not PC12 cells. Jurkat cells (A) and PC12 cells (B) were preincubated with PBS (CON), 20 µM genistein (Gen), or 35 µM MLA for 60 min and then loaded with indo-1. After washing, cells were exposed to 200 µM nicotine, and [Ca2+]i was determined by fluorometry as described in Materials and Methods. Results are representative of two replicates from two independent experiments.

View larger version (14K): [in this window] [in a new window]   FIGURE 5. Nicotine and MLA raise [Ca2+]i in Jurkat cells. Indo-1-loaded Jurkat cells were preincubated in PBS alone (A) or 35 µM MLA (B), and changes in [Ca2+]i were determined in response to 100 µM nicotine. The baseline Ca2+ level after MLA pretreatment was usually 100–200 nM higher than PBS-treated control. Some indo-1-loaded Jurkat cells were first treated with 35 µM MLA and followed by the addition of 100 µM nicotine at 600 s after the MLA addition (C). Results are representative of two to three independent experiments.

Activation of T cells through the TCR is initiated through an early wave of protein tyrosine phosphorylation mediated by the Src kinases Lck and Fyn, leading to the activation of downstream signaling, including an increase in [Ca²⁺]_i from release of Ca²⁺ from intracellular Ca²⁺ stores and Ca²⁺-induced Ca²⁺ influx (45). To ascertain whether nicotine-activated PTK, WT, and Lck^– Jurkat cells were treated with 1 µM nicotine or 5 µg/ml anti-TCR Ab for 10 min. Cell extracts were resolved on SDS gels and probed with anti-phosphotyrosine Ab. Although both nicotine and anti-TCR Ab activated a similar PTK profile in WT Jurkat cells, they did not activate PTK activity in Lck^– Jurkat cells (Fig. 6). These results suggest that activation of PTK by nicotine in T cells closely resembles the activation of T cells via the TCR, and both require functional Lck.

View larger version (101K): [in this window] [in a new window]   FIGURE 6. Anti-TCR and nicotine activate PTKs in WT (original) but not Lck– Jurkat cells. WT and Lck– Jurkat cells were incubated with anti-TCR Abs (-TCR) or nicotine (NT) for 10 min. Twenty micrograms of protein of cell extracts were run on SDS-PAGE and developed with anti-phosphotyrosine Abs as described in Materials and Methods. Control (CON) samples were from cells incubated in the absence of nicotine or anti-TCR Abs. Results are representative of two independent experiments.

View larger version (49K): [in this window] [in a new window]   FIGURE 7. Nicotine activates Lck and Fyn activities in WT but not Lck– Jurkat cells. Jurkat cells (WT and Lck–) were incubated with (+) and without (–) 1 µM nicotine for 10 min. Cell extracts equivalent to 150 µg of protein were immunoprecipitated with anti-Lck or Fyn Abs. (A) Lck activity (autophosphorylation) and (B) Fyn (enolase phosphorylation) activities were determined in the immunoprecipitates as described in Materials and Methods. The results are representative of three replicate samples from two independent experiments.

Lck has been implicated in the TCR-mediated increase in [Ca²⁺]_i response (46). Because nicotine activates Lck, we determined whether Lck was required for the nicotine-induced Ca²⁺ response in T cells. Interestingly, while nicotine treatment and TCR ligation with anti-TCR Abs increased [Ca²⁺]_i in WT Jurkat cells, neither nicotine nor anti-TCR increased [Ca²⁺]_i significantly in TCR/CD3-β^– or Lck^– Jurkat cell lines (Fig. 8). These results suggest that nicotine and TCR have similar signaling requirements for increasing the [Ca²⁺]_i in T cells, and the nicotine-induced rise in [Ca²⁺]_i requires a functionally intact TCR/CD3 complex and Lck.

View larger version (17K): [in this window] [in a new window]   FIGURE 8. Nicotine and anti-TCR fail to raise significant [Ca2+]i from β– and Lck– Jurkat cells. Indo-1-loaded cells from WT, β–, and Lck– Jurkat cells were exposed to 5 µg/ml anti-TCR (upper panels) or 100 µM nicotine (NT) (lower panels), and [Ca2+]i was determined as described in Materials and Methods. The results are representative of two replicate samples from two independent experiments.

Knockdown of 7-nAChR suppresses nicotine but not the anti-TCR-mediated increase in [Ca²⁺]_i

We examined whether the similarities between Ag- and nicotine-induced Ca²⁺ signaling in T cells might result from direct activation of the TCR with nicotine that is independent of 7-nAChRs. WT Jurkat cells were treated with 7-nAChR-specific antisense RNA to knockdown 7-nAChR subunit expression. Antisense RNA was introduced through electroporation, and the treatment caused a significant decrease in the mRNA (Fig. 9A, top panel) and protein (Fig. 9A, lower panel) densities of the 7-nAChR subunit (Fig. 9A, top panel). Moreover, compared with electroporated control (sham-treated) cells, the ability of antisense RNA-treated cells to bind -BTX was significantly reduced (Fig. 9B), and following nicotine treatment, the antisense RNA-treated cells exhibited significantly reduced rise in [Ca²⁺]_i than untreated (CON) or sham-treated (sham) cells (Fig. 9C). However, 7-nAChR subunit-deficient (antisense RNA-treated) Jurkat cells exhibited a normal TCR-induced Ca²⁺ response (Fig. 9D). Similar results were observed when 7-nAChR subunit mRNA was knocked down with siRNA in purified rat splenic T cells (data not shown). Thus, in T cells, 7-nAChRs are important for the nicotine-induced, but not the TCR-mediated, rise in [Ca²⁺]_i.

View larger version (33K): [in this window] [in a new window]   FIGURE 9. Antisense 7-nAChR RNA down-regulates 7-nAChR expression and the nicotine-induced Ca2+ response in Jurkat cells. Jurkat cells were treated with 7-nAChR antisense RNA or electroporated without the RNA (sham). At 36 h after electroporation untreated (CON), sham, and antisense-treated cells were compared for A: 7-nAChR and GAPDH mRNA expression by RT-PCR (A, upper panel) and 7-nAChR protein and actin expression by Western blot analysis of 10 µg of cell lysate proteins (A, lower panel). B, Binding of Alexa Fluor 488-labeled -BTX by sham- and antisense RNA-treated cells was examined by flow cytometry, where unlabeled sham-treated cells provided background (BK) fluorescence values for the cells. At least 10,000 cells were examined for each run. C, Changes in [Ca2+]i of the cells in response to 100 µM nicotine were determined by fluorometry as described in Fig. 4. D, Changes in [Ca2+]i in response to anti-TCR/second Ab were determined by spectrofluorometry as described Fig. 4.

CD3 and 7-nAChR coprecipitate

Because 7-nAChR subunits appear to use the TCR-associated signaling components to raise [Ca²⁺]_i in T cells in response to nicotine, we determined whether 7-nAChR subunits were in close proximity to the TCR/CD3 complex and interacted with each other. Cell extracts from WT Jurkat cells were immunoprecipitated with either the isotype control IgG, 7-nAChR-specific polyclonal Ab, or anti-CD3 chain-specific mAb. The immunoprecipitates were resolved on PAGE and probed with either an anti-CD3 chain-specific mAb or anti-7-nAChR Abs. Results indicate that the 7-nAChR subunit and CD3 coprecipitate with either of the two Abs, suggesting that the two receptor proteins interact with each other in T cells (Fig. 10).

View larger version (36K): [in this window] [in a new window]   FIGURE 10. CD3 and 7-nAChRs coimmunoprecipitate. Jurkat cell lysates were treated with anti-CD3, anti-7-nAChR, isotype control Ig, or protein A beads. The immunoprecipitates (IP) were captured by protein A beads. The lysates, immunoprecipitates, lysates treated with isotype control Ab plus beads, and lysates treated only with beads were resolved by electrophoresis, blotted, and the blots were probed with anti-CD3 chain-specific (upper panels) or anti-7-nAChR (lower panels) mAbs as described in Materials and Methods.

To rule out the possibility that in T cells a small but significant initial influx of Ca²⁺ through the purported nAChR channels might subsequently trigger a larger second messenger-driven Ca²⁺ response, Jurkat cells were stimulated with nicotine in calcium-free medium containing 5 mM EGTA. Results presented in Fig. 11 clearly show that even in the absence of outside Ca²⁺, both nicotine and anti-TCR raise [Ca²⁺]_i through the release of calcium from internal stores, and this rise is quenched by preincubation of cells with the Ca²⁺ chelator BAPTA (4 µM). Thus, mobilization of Ca²⁺ through a putative nAChR is not required for the nicotine-induced release of Ca²⁺ from the internal stores. This indicates that the initial rise in [Ca²⁺]_i in T cells in response to nicotine is independent of external Ca²⁺ influx. Therefore, it is unlikely that 7-nAChR subunits form functional nicotine-sensitive cation channels in T cells.

View larger version (28K): [in this window] [in a new window]   FIGURE 11. Extracellular Ca2+ is not required for the release of Ca2+ from the intracellular Ca2+ stores in response to nicotine and anti-TCR. Indo-1-loaded Jurkat cells were treated with 1 mM nicotine (NT) or anti-TCR Abs (TCR) in Ca2+-containing medium (top panels), in 5 mM EGTA-containing Ca2+-free medium (middle panels), or treated with BAPTA during indo-1 loading and then transferred into EGTA-containing Ca2+-free medium (bottom panels). Changes in [Ca2+]i were assayed by fluorometry as described in Fig. 4.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

Besides increasing [Ca²⁺]_i, our results clearly show that stimulation of T cells with nicotine induces PTK activities, including an increased activity of the Src kinases, Fyn and Lck. In T cells, the TCR-induced rise in [Ca²⁺]_i is initiated with the activation of Lck, Fyn, and the -associated protein kinase, leading to the activation of downstream signaling pathways (49, 50). Because activation of PTKs is critical for the TCR-induced Ca²⁺ response, we determined whether it was important in the nicotine-induced increase in [Ca²⁺]_i. Surprisingly, preincubation of Jurkat cells with the tyrosine kinase inhibitor genistein blocked the TCR-mediated Ca²⁺ response and suppressed the nicotine-induced rise in [Ca²⁺]_i. In contrast, genistein did not significantly affect the rise in [Ca²⁺]_i in PC12 cells. This finding is further supported by the results that Lck^– Jurkat cells were unable to significantly raise [Ca²⁺]_i in response to nicotine or anti-TCR Abs. Therefore, unlike neuronal cells, PTKs play an important role in the nicotine-induced Ca²⁺ response in T cells; this response uses the signaling pathway similar to that triggered by an Ag in T cells.

In neurons, nAChRs are recognized as Ca²⁺-permeating cation channels, so it was surprising to find that the Ca²⁺ response stimulated by nicotine in T cells is tightly associated with the activation of PTKs (i.e., second messenger-activated response) and not through direct influx of Ca²⁺ through a nAChR channel. It is conceivable, although unlikely, that the increase in [Ca²⁺]_i by nicotine in T cells resulted from some spurious activation of the TCR by nicotine that was independent of nAChRs. However, in Jurkat cells, down-regulation of the 7-nAChR subunit expression by RNA interference resulted in a significant reduction in the nicotine-induced, but not in the TCR-induced, rise in [Ca²⁺]_i. Although these results do not eliminate the possibility that non-7-nAChRs may contribute to the nicotine-induced Ca²⁺ response in T cells, the results strongly suggest that the 7-nAChR subunit plays a predominant role in this response, and thus it is unlikely that nicotine interacts directly with TCR to raise [Ca²⁺]_i. In contrast, preliminary coimmunoprecipitation experiments suggest that the 7-nAChR subunit protein and TCR might interact with each other in a manner such that binding of nicotine to 7-nAChRs activates the TCR and its signaling machinery to stimulate the Ca²⁺ response. The observation that the -chain-deficient TCR/CD3 complex is ineffective in transmitting signals to raise [Ca²⁺]_i in response to nicotine further supports this inference.

-BTX and MLA are considered relatively specific antagonists of 7-nAChRs and block nicotine-induced currents in neuronal cells (16). However, contrary to their action in PC12 cells, both -BTX and MLA stimulated a strong Ca²⁺ response in T cells. Therefore, these 7-nAChR antagonists in neuronal cells act as agonists of the receptor in T cells. Sharma and Vijayaraghavan (32, 33) have also observed that activation of 7-nAChRs in astrocytes causes Ca²⁺ influx and a potent second messenger response, which in turn amplifies [Ca²⁺]_i. The role of Ca²⁺ release-activated Ca²⁺ channels in a TCR-derived Ca²⁺ response has been well-documented (51, 52, 53). Therefore, it is possible that a small, essentially undetectable amount of Ca²⁺ influx through the 7-nAChR subunit-forming channel is required for PTK activation and the release of calcium from IP3-sensitive calcium stores. The Ca²⁺ released from these stores would then trigger Ca²⁺ influx from Ca²⁺ release-activated Ca²⁺ channels to further amplify the level of [Ca²⁺]_i (51). To ascertain this possibility, Jurkat cells were treated with nicotine in EGTA-containing Ca²⁺-free medium to prevent the entry of Ca²⁺. Our results clearly show that, in the total absence of extracellular Ca²⁺, nicotine released Ca²⁺ from the intracellular Ca²⁺ stores, and this increase in [Ca²⁺]_i was quenched by BAPTA. Furthermore, our recent experiments also indicate that the nicotine-induced Ca²⁺ release from intracellular stores is dependent on PTK activation (our unpublished observation). Thus, it is unlikely that in T cells the nicotine-stimulated rise in [Ca²⁺]_i is regulated by nAChRs that form ligand-gated Ca²⁺ channels. Given that T cells express 7-nAChR subunits essentially identical to the neuronal receptor, failure to detect a functional Ca²⁺-permeating channel is surprising. However, even in some PC12 cells and TsA201 cells transfected with 7-nAChR cDNA, the presence of full-length 7-nAChR mRNA transcripts and membrane-localized 7-nAChRs was not sufficient to generate a functional nicotine-sensitive ligand-gated Ca²⁺ response (31, 34). RIC-3, a chaperon protein, has recently been implicated in the functional association of 7-nAChRs (54). Our preliminary experiments indicate that ric-3 mRNA is expressed in normal (WT) and Lck^– Jurkat T cells (our unpublished observation) and has a 99% sequence homology with neuronal ric-3 in a stretch of 1568 nucleotides (GenBank accession number for WT Jurkat: DQ 347704 and for Lck^–: DQ 347704). Nonetheless, it is possible that other hitherto unidentified proteins, absent in T cells, facilitate assembly and stabilization of 7-nAChRs in neurons. Our experiments clearly show that, if nicotine-sensitive ligand-gated cation channels exist on T cells, these channels are not essential for the nicotine-induced rise in [Ca²⁺]_i. In contrast, 7-nAChR subunits interact with the TCR and raise [Ca²⁺]_i through the TCR-associated signaling pathway. Whether a similar mechanism for elevating [Ca²⁺]_i in response to nicotine operates in other nonexcitable cells is unknown at the present time.

	Acknowledgments

 
We thank Dr. S. Vijayaraghavan (University of Colorado Health Sciences Center, Denver, CO) for technical advice. We also thank Paula Bradley and Vicki Fisher for editorial help.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported in part by grants from the National Institutes of Health (RO1 DA017003, R01 DA04208-15, and RO1DA04208).

² Address correspondence and reprint requests to Dr. Mohan L. Sopori, Lovelace Respiratory Research Institute, 2425 Ridgecrest Drive SE, Albuquerque, NM 87108. E-mail address: msopori{at}lrri.org

³ Abbreviations used in this paper: nAChR, nicotinic acetylcholine receptor; -BTX, -bungarotoxin; MLA, methyllycaconitine; [Ca²⁺]_i, intracellular calcium; PTK, protein tyrosine kinase; WT, wild type; hT, human T cell; [³H]EB, [³H]epibatidine; Lck, leukocyte-specific tyrosine kinase; siRNA, small interfering RNA.

Received for publication November 29, 2006. Accepted for publication June 21, 2007.

	References

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1. Role, L. W., D. K. Berg. 1996. Nicotinic receptors in the development and modulation of CNS synapses. Neuron 16: 1077-1085. [Medline]
2. MacDermott, A. B., L. W. Role, S. A. Siegelbaum. 1999. Presynaptic ionotropic receptors and the control of transmitter release. Annu. Rev. Neurosci. 22: 443-485. [Medline]
3. Vijayaraghavan, S., B. Huang, E. M. Blumenthal, D. K. Berg. 1995. Arachidonic acid as a possible negative feedback inhibitor of nicotinic acetylcholine receptors on neurons. J. Neurosci. 15: 3679-3687. [Abstract]
4. Vernallis, A. B., W. G. Conroy, D. K. Berg. 1993. Neurons assemble acetylcholine receptors with as many as three kinds of subunits while maintaining subunit segregation among receptor subtypes. Neuron 10: 451-464. [Medline]
5. Lindstrom, J. M.. 2003. Nicotinic acetylcholine receptors of muscles and nerves: comparison of their structures, functional roles, and vulnerability to pathology. Ann. NY Acad. Sci. 998: 41-52. [Medline]
6. Gotti, C., F. Clementi. 2004. Neuronal nicotinic receptors: from structure to pathology. Prog. Neurobiol. 74: 363-396. [Medline]
7. Ullian, E. M., J. M. McIntosh, P. B. Sargent. 1997. Rapid synaptic transmission in the avian ciliary ganglion is mediated by two distinct classes of nicotinic receptors. J. Neurosci. 17: 7210-7219. [Abstract/Free Full Text]
8. Chang, K. T., D. K. Berg. 1999. Nicotinic acetylcholine receptors containing 7 subunits are required for reliable synaptic transmission in situ. J. Neurosci. 19: 3701-3710. [Abstract/Free Full Text]
9. Millar, N. S.. 2003. Assembly and subunit diversity of nicotinic acetylcholine receptors. Biochem. Soc. Trans. 31: 869-874. [Medline]
10. Tomizawa, M., J. E. Casida. 2003. Selective toxicity of neonicotinoids attributable to specificity of insect and mammalian nicotinic receptors. Annu. Rev. Entomol. 48: 339-364. [Medline]
11. Lansdell, S. J., N. S. Millar. 2004. Molecular characterization of D6 and D7 nicotinic acetylcholine receptor subunits from Drosophila: formation of a high-affinity -bungarotoxin binding site revealed by expression of subunit chimeras. J. Neurochem. 90: 479-489. [Medline]
12. Arredondo, J., V. T. Nguyen, A. I. Chernyavsky, D. Bercovich, A. Orr-Urtreger, W. Kummer, K. Lips, D. E. Vetter, S. A. Grando. 2002. Central role of 7 nicotinic receptor in differentiation of the stratified squamous epithelium. J. Cell Biol. 159: 325-336. [Abstract/Free Full Text]
13. Wang, Y., E. F. Pereira, A. D. Maus, N. S. Ostlie, D. Navaneetham, S. Lei, E. X. Albuquerque, B. M. Conti-Fine. 2001. Human bronchial epithelial and endothelial cells express 7 nicotinic acetylcholine receptors. Mol. Pharmacol. 60: 1201-1209. [Abstract/Free Full Text]
14. Razani-Boroujerdi, S., M. L. Sopori. 2007. Early manifestations of NNK-induced lung cancer: role of lung immunity in tumor susceptibility. Am. J. Respir. Cell. Mol. Biol. 36: 13-19. [Abstract/Free Full Text]
15. Saeed, R. W., S. Varma, T. Peng-Nemeroff, B. Sherry, D. Balakhaneh, J. Huston, K. J. Tracey, Y. Al Abed, C. N. Metz. 2005. Cholinergic stimulation blocks endothelial cell activation and leukocyte recruitment during inflammation. J. Exp. Med. 201: 1113-1123. [Abstract/Free Full Text]
16. Nai, Q., J. M. McIntosh, J. F. Margiotta. 2003. Relating neuronal nicotinic acetylcholine receptor subtypes defined by subunit composition and channel function. Mol. Pharmacol. 63: 311-324. [Abstract/Free Full Text]
17. Kulak, J. M., F. I. Carroll, J. S. Schneider. 2006. [¹²⁵I]Iodomethyllycaconitine binds to 7 nicotinic acetylcholine receptors in monkey brain. Eur. J. Neurosci. 23: 2604-2610. [Medline]
18. Sopori, M. L., W. Kozak, S. M. Savage, Y. Geng, M. J. Kluger. 1998. Nicotine-induced modulation of T cell function: implications for inflammation and infection. Adv. Exp. Med. Biol. 437: 279-289. [Medline]
19. Sopori, M.. 2002. Effects of cigarette smoke on the immune system. Nat. Rev. Immunol. 2: 372-377. [Medline]
20. Wang, H., M. Yu, M. Ochani, C. A. Amella, M. Tanovic, S. Susarla, J. H. Li, H. Wang, H. Yang, L. Ulloa, et al 2003. Nicotinic acetylcholine receptor 7 subunit is an essential regulator of inflammation. Nature 421: 384-388. [Medline]
21. Geng, Y., S. M. Savage, S. Razani-Boroujerdi, M. L. Sopori. 1996. Effects of nicotine on the immune response. II. Chronic nicotine treatment induces T cell anergy. J. Immunol. 156: 2384-2390. [Abstract]
22. Yong, T., M. Q. Zheng, D. S. Linthicum. 1997. Nicotine induces leukocyte rolling and adhesion in the cerebral microcirculation of the mouse. J. Neuroimmunol. 80: 158-164. [Medline]
23. Shin, V. Y., W. K. Wu, K. M. Chu, H. P. Wong, E. K. Lam, E. K. Tai, M. W. Koo, C. H. Cho. 2005. Nicotine induces cyclooxygenase-2 and vascular endothelial growth factor receptor-2 in association with tumor-associated invasion and angiogenesis in gastric cancer. Mol. Cancer Res. 3: 607-615. [Abstract/Free Full Text]
24. Czura, C. J., K. J. Tracey. 2005. Autonomic neural regulation of immunity. J. Intern. Med. 257: 156-166. [Medline]
25. Battaglioli, E., C. Gotti, S. Terzano, A. Flora, F. Clementi, D. Fornasari. 1998. Expression and transcriptional regulation of the human 3 neuronal nicotinic receptor subunit in T lymphocyte cell lines. J. Neurochem. 71: 1261-1270. [Medline]
26. Lustig, L. R., H. Peng, H. Hiel, T. Yamamoto, P. A. Fuchs. 2001. Molecular cloning and mapping of the human nicotinic acetylcholine receptor 10 (CHRNA10). Genomics 73: 272-283. [Medline]
27. Kuo, Y., L. Lucero, J. Michaels, D. DeLuca, R. J. Lukas. 2002. Differential expression of nicotinic acetylcholine receptor subunits in fetal and neonatal mouse thymus. J. Neuroimmunol. 130: 140-154. [Medline]
28. Kimura, R., N. Ushiyama, T. Fujii, K. Kawashima. 2003. Nicotine-induced Ca²⁺ signaling and down-regulation of nicotinic acetylcholine receptor subunit expression in the CEM human leukemic T-cell line. Life Sci. 72: 2155-2158. [Medline]
29. Peng, H., R. L. Ferris, T. Matthews, H. Hiel, A. Lopez-Albaitero, L. R. Lustig. 2004. Characterization of the human nicotinic acetylcholine receptor subunit () 9 (CHRNA9) and () 10 (CHRNA10) in lymphocytes. Life Sci. 76: 263-280. [Medline]
30. Shytle, R. D., T. Mori, K. Townsend, M. Vendrame, N. Sun, J. Zeng, J. Ehrhart, A. A. Silver, P. R. Sanberg, J. Tan. 2004. Cholinergic modulation of microglial activation by 7 nicotinic receptors. J. Neurochem. 89: 337-343. [Medline]
31. Blumenthal, E. M., W. G. Conroy, S. J. Romano, P. D. Kassner, D. K. Berg. 1997. Detection of functional nicotinic receptors blocked by -bungarotoxin on PC12 cells and dependence of their expression on post-translational events. J. Neurosci. 17: 6094-6104. [Abstract/Free Full Text]
32. Sharma, G., S. Vijayaraghavan. 2001. Nicotinic cholinergic signaling in hippocampal astrocytes involves calcium-induced calcium release from intracellular stores. Proc. Natl. Acad. Sci. USA 98: 4148-4153. [Abstract/Free Full Text]
33. Sharma, G., S. Vijayaraghavan. 2002. Nicotinic receptor signaling in nonexcitable cells. J. Neurobiol. 53: 524-534. [Medline]
34. Rakhilin, S., R. C. Drisdel, D. Sagher, D. S. McGehee, Y. Vallejo, W. N. Green. 1999. -Bungarotoxin receptors contain 7 subunits in two different disulfide-bonded conformations. J. Cell Biol. 146: 203-218. [Abstract/Free Full Text]
35. Williams, M. E., B. Burton, A. Urrutia, A. Shcherbatko, L. E. Chavez-Noriega, C. J. Cohen, J. Aiyar. 2005. Ric-3 promotes functional expression of the nicotinic acetylcholine receptor 7 subunit in mammalian cells. J. Biol. Chem. 280: 1257-1263. [Abstract/Free Full Text]
36. Lansdell, S. J., V. J. Gee, P. C. Harkness, A. I. Doward, E. R. Baker, A. J. Gibb, N. S. Millar. 2005. RIC-3 enhances functional expression of multiple nicotinic acetylcholine receptor subtypes in mammalian cells. Mol. Pharmacol. 68: 1431-1438. [Abstract/Free Full Text]
37. Davila-Garcia, M. I., J. L. Musachio, D. C. Perry, Y. Xiao, A. Horti, E. D. London, R. F. Dannals, K. J. Kellar. 1997. [¹²⁵I]IPH, an epibatidine analog, binds with high affinity to neuronal nicotinic cholinergic receptors. J. Pharmacol. Exp. Ther. 282: 445-451. [Abstract/Free Full Text]
38. Razani-Boroujerdi, S., L. D. Partridge, M. L. Sopori. 1994. Intracellular calcium signaling induced by thapsigargin in excitable and inexcitable cells. Cell Calcium 16: 467-474. [Medline]
39. Xiao, Y., K. J. Kellar. 2004. The comparative pharmacology and up-regulation of rat neuronal nicotinic receptor subtype binding sites stably expressed in transfected mammalian cells. J. Pharmacol. Exp. Ther. 310: 98-107. [Abstract/Free Full Text]
40. Peng, J. H., J. D. Fryer, R. S. Hurst, K. M. Schroeder, A. A. George, S. Morrissy, V. E. Groppi, S. S. Leonard, R. J. Lukas. 2005. High-affinity epibatidine binding of functional, human 7-nicotinic acetylcholine receptors stably and heterologously expressed de novo in human SH-EP1 cells. J. Pharmacol. Exp. Ther. 313: 24-35. [Abstract/Free Full Text]
41. Stitzel, J. A., D. A. Farnham, A. C. Collins. 1996. Linkage of strain-specific nicotinic receptor 7 subunit restriction fragment length polymorphisms with levels of -bungarotoxin binding in brain. Brain Res. Mol. Brain Res. 43: 30-40. [Medline]
42. Weiland, S., D. Bertrand, S. Leonard. 2000. Neuronal nicotinic acetylcholine receptors: from the gene to the disease. Behav. Brain Res. 113: 43-56. [Medline]
43. Couturier, S., D. Bertrand, J. M. Matter, M. C. Hernandez, S. Bertrand, N. Millar, S. Valera, T. Barkas, M. Ballivet. 1990. A neuronal nicotinic acetylcholine receptor subunit (7) is developmentally regulated and forms a homo-oligomeric channel blocked by -BTX. Neuron 5: 847-856. [Medline]
44. Seguela, P., J. Wadiche, K. Dineley-Miller, J. A. Dani, J. W. Patrick. 1993. Molecular cloning, functional properties, and distribution of rat brain 7: a nicotinic cation channel highly permeable to calcium. J. Neurosci. 13: 596-604. [Abstract]
45. Nel, A. E.. 2002. T-cell activation through the antigen receptor. Part 1. Signaling components, signaling pathways, and signal integration at the T-cell antigen receptor synapse. J. Allergy Clin. Immunol. 109: 758-770. [Medline]
46. Methi, T., J. Ngai, M. Mahic, M. Amarzguioui, T. Vang, K. Tasken. 2005. Short-interfering RNA-mediated Lck knockdown results in augmented downstream T cell responses. J. Immunol. 175: 7398-7406. [Abstract/Free Full Text]
47. Bertrand, D., J. L. Galzi, A. Devillers-Thiery, S. Bertrand, J. P. Changeux. 1993. Mutations at two distinct sites within the channel domain M2 alter calcium permeability of neuronal 7 nicotinic receptor. Proc. Natl. Acad. Sci. USA 90: 6971-6975. [Abstract/Free Full Text]
48. Charpantier, E., A. Wiesner, K. H. Huh, R. Ogier, J. C. Hoda, G. Allaman, M. Raggenbass, D. Feuerbach, D. Bertrand, C. Fuhrer. 2005. 7 neuronal nicotinic acetylcholine receptors are negatively regulated by tyrosine phosphorylation and Src-family kinases. J. Neurosci. 25: 9836-9849. [Abstract/Free Full Text]
49. Weiss, A., D. R. Littman. 1994. Signal transduction by lymphocyte antigen receptors. Cell 76: 263-274. [Medline]
50. Nel, A. E., N. Slaughter. 2002. T-cell activation through the antigen receptor. Part 2. role of signaling cascades in T-cell differentiation, anergy, immune senescence, and development of immunotherapy. J. Allergy Clin. Immunol. 109: 901-915. [Medline]
51. Parekh, A. B., J. W. Putney, Jr. 2005. Store-operated calcium channels. Physiol. Rev. 85: 757-810. [Abstract/Free Full Text]
52. Prakriya, M., R. S. Lewis. 2003. CRAC channels: activation, permeation, and the search for a molecular identity. Cell. Calcium 33: 311-321. [Medline]
53. Panyi, G., Z. Varga, R. Gaspar. 2004. Ion channels and lymphocyte activation. Immunol. Lett. 92: 55-66. [Medline]
54. Ben Ami, H. C., L. Yassin, H. Farah, A. Michaeli, M. Eshel, M. Treinin. 2005. RIC-3 affects properties and quantity of nicotinic acetylcholine receptors via a mechanism that does not require the coiled-coil domains. J. Biol. Chem. 280: 28053-28060. [Abstract/Free Full Text]

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