Nicotine-induced Ca2+ signaling and down-regulation of nicotinic acetylcholine receptor subunit expression in the CEM human leukemic T-cell line (original) (raw)
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The Journal of Immunology, 2007
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 ([Ca 2؉ ] 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 [Ca 2؉ ] 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 Ca 2؉ response, but unlike the neuronal receptor, ␣-bungarotoxin and methyllycaconitine not only failed to block, but also actually raised [Ca 2؉ ] i in T cells. The nicotine-induced release of Ca 2؉ from intracellular stores in T cells did not require extracellular Ca 2؉ , but, similar to the TCR-mediated Ca 2؉ 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 Ca 2؉ channel, and that the nicotineinduced rise in [Ca 2؉ ] i in T cells requires functional TCR/CD3 and leukocyte-specific tyrosine kinase.
The Journal of …, 2007
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 ([Ca 2؉ ] 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 [Ca 2؉ ] 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 Ca 2؉ response, but unlike the neuronal receptor, ␣-bungarotoxin and methyllycaconitine not only failed to block, but also actually raised [Ca 2؉ ] i in T cells. The nicotine-induced release of Ca 2؉ from intracellular stores in T cells did not require extracellular Ca 2؉ , but, similar to the TCR-mediated Ca 2؉ 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 Ca 2؉ channel, and that the nicotineinduced rise in [Ca 2؉ ] i in T cells requires functional TCR/CD3 and leukocyte-specific tyrosine kinase.
Alpha 7 nicotinic acetylcholine receptor modulates lymphocyte activation
Life Sciences, 2009
Even though the presence of α7 nicotinic receptor (nAChR) in lymphocytes has been demonstrated, its functional role still remains elusive. The aim of our study was to characterize α7 nAChRs in human lymphocytes upon phytohemagglutinin (PHA) stimulation. Main methods: Lymphocytes were activated with the mitogen PHA. α7 nAChRs were studied by reverse transcription-polymerase chain reaction (RT-PCR), real time PCR, flow cytometry and confocal laser scanning microscopy. The effects of nicotinic drugs on PHA-induced proliferation was evaluated by the [ 3 H]-thymidine incorporation assay. Key findings: We show that the expression of functional α7 receptors increases after PHA stimulation. The activation of peripheral lymphocytes by PHA increases 2.2-fold the α7 subunit mRNA expression and 4-fold the binding of the antagonist α-bungarotoxin (α-BTX) with respect to non activated lymphocytes. By measuring the increase of intracellular calcium in response to nicotine we determine that α7 receptors in lymphocytes are functional. Nicotinic drugs differentially modulate T cell activation. While nicotine tends to inhibit proliferative responses, specific α7 antagonists, such as α-BTX and methyllycaconitine, enhance cell division. Significance: This study reveals that the α7 receptor modulates lymphocyte activation and contributes to clarifying the role of the non neuronal cholinergic system in the immune response.
The Journal of Immunology, 2010
Smokers are less likely to develop some inflammatory and allergic diseases. In Brown-Norway rats, nicotine inhibits several parameters of allergic asthma, including the production of Th2 cytokines and the cysteinyl leukotriene LTC 4 . Cysteinyl leukotrienes are primarily produced by mast cells, and these cells play a central role in allergic asthma. Mast cells express a highaffinity receptor for IgE (Fc«RI). Following its cross-linking, cells degranulate and release preformed inflammatory mediators (early phase) and synthesize and secrete cytokines/chemokines and leukotrienes (late phase). The mechanism by which nicotine modulates mast cell activation is unclear. Using a-bungarotoxin binding and quantitative PCR and PCR product sequencing, we showed that the rat mast/basophil cell line RBL-2H3 expresses nicotinic acetylcholine receptors (nAChRs) a7, a9, and a10; exposure to exceedingly low concentrations of nicotine (nanomolar), but not the biologically inactive metabolite cotinine, for ‡8 h suppressed the late phase (leukotriene/cytokine production) but not degranulation (histamine and hexosaminidase release). These effects were unrelated to those of nicotine on intracellular free calcium concentration but were causally associated with the inhibition of cytosolic phospholipase A 2 activity and the PI3K/ERK/NF-kB pathway, including phosphorylation of Akt and ERK and nuclear translocation of NF-kB. The suppressive effect of nicotine on the late-phase response was blocked by the a7/a9-nAChR antagonists methyllycaconitine and a-bungarotoxin, as well as by small interfering RNA knockdown of a7-, a9-, or a10-nAChRs, suggesting a functional interaction between a7-, a9-, and a10-nAChRs that might explain the response of RBL cells to nanomolar concentrations of nicotine. This "hybrid" receptor might serve as a target for novel antiallergic/antiasthmatic therapies.
Journal of Biological Chemistry, 2004
Desensitization induced by chronic nicotine exposure has been hypothesized to trigger the up-regulation of the ␣42 neuronal nicotinic acetylcholine receptor (nAChR) in the central nervous system. We studied the effect of acute and chronic nicotine exposure on the desensitization and up-regulation of different ␣42 subunit ratios (1␣:4, 2␣:3, and 4␣:1) expressed in Xenopus oocytes. The presence of ␣4 subunit in the oocyte plasmatic membrane increased linearly with the amount of ␣4 mRNA injected. nAChR function and expression were assessed during acute and after chronic nicotine exposure using a two-electrode voltage clamp and whole-mount immunofluorescence assay along with confocal imaging for the detection of the ␣4 subunit. The 2␣4:32 subunit ratio displayed the highest ACh sensitivity. Nicotine doseresponse curves for the 1␣4:42 and 2␣4:32 subunit ratios displayed a biphasic behavior at concentrations ranging from 0.1 to 300 M. A biphasic curve for 4␣4:12 was obtained at nicotine concentrations higher than 300 M. The 1␣4:42 subunit ratio exhibited the lowest ACh-and nicotine-induced macroscopic current, whereas 4␣4:12 presented the largest currents at all agonist concentrations tested. Desensitization by acute nicotine exposure was more evident as the ratio of 2:␣4 subunits increased. All three ␣42 subunit ratios displayed a reduced state of activation after chronic nicotine exposure. Chronic nicotine-induced up-regulation was obvious only for the 2␣4: 32 subunit ratio. Our data suggest that the subunit ratio of ␣42 determines the functional state of activation, desensitization, and up-regulation of this neuronal nAChR. We propose that independent structural sites regulate ␣42 receptor activation and desensitization.
The FASEB Journal, 2007
Tobacco products and nicotine alter the cell cycle and lead to squamatization of oral keratinocytes (KCs) and squamous cell carcinoma. Activation of nicotinic acetylcholine receptors (nAChRs) elicits Ca 2؉ influx that varies in magnitude between different nAChR subtypes. Normal differentiation of KCs is associated with sequential expression of the nAChR subtypes with increasing Ca 2؉ permeability, such as ␣5-containing ␣3 nAChR and ␣7 nAChR. Exposure to environmental tobacco smoke (ETS) or an equivalent concentration of nicotine accelerated by severalfold the ␣5 and ␣7 expression in KCs, which could be abolished by mecamylamine and ␣-bungarotoxin with different efficacies, suggesting the following sequence of autoregulation of the expression of nAChR subtypes: ␣3(2/4) > ␣3(2/4)␣5 > ␣7 > ␣7. This conjecture was corroborated by results of quantitative assays of subunit mRNA and protein levels, using nAChRspecific pharmacologic antagonists and small interfering RNAs. The genomic effects of ETS and nicotine involved the transcription factor GATA-2 that showed a multifold increase in quantity and activity in exposed KCs. Using protein kinase inhibitors and dominant negative and constitutively active constructs, we characterized the principal signaling cascades mediating a switch in the nAChR subtype. Cumulative results indicated that the ␣3(2/4) to ␣3(2/4)␣5 nAChR transition predominantly involved protein kinase C, ␣3(2/ 4)␣5 to ␣7 nAChR transition-Ca 2؉ /calmodulindependent protein kinase II and p38 MAPK, and ␣7 self-up-regulation-the p38 MAPK/Akt pathway, and JAK-2. These results provide a mechanistic insight into the genomic effects of ETS and nicotine on KCs and characterize signaling pathways mediating autoregulation of stepwise overexpression of nAChR subtypes with increasing Ca 2؉ permeability in exposed cells. These observations have salient clinical implications, because a switch in the nAChR subunit composition can bring about a corresponding switch in receptor function, leading to profound pathobiologic effects observed in KCs exposed to tobacco products. Arre
Nicotine and acetylcholine lead to distinct modulation of gene regulation
Background: Sepsis is well known to lead to the activation of multiple pro-inflammatory markers, like MCP-1 (Monocyte chemotactic protein 1), TNF-alpha (Tumor necrosis factor alpha), while the underlying genetic changes still remain poorly studied.Methods: We used human umbilical vein endothelial cells to test the reactions to nicotine or acetylcholine/pyridostigmine administration in regards to MCP-1 levels, gene regulation and RNR expression. Results: Pyridostigmine and Acetylcholine (Ach) lead to a significant decrease of MCP-1 levels in TNF-alpha stimulated human umbilical vein endothelial cells, while nicotine had no effect. Interestingly nicotine and acetylcholine lead to different gene expression (nicotine up-regulates epidermal growth factor and down-regulates matrix metalloproteinase-8, while Ach/pyridostigmine up-regulates thioredoxin interacting protein and down-regulates insulin like growth factor 1). Furthermore RNA levels and gene activation were similar after nicotine...