title: Nitric oxide (NO) synthase mRNA expression and NO production via muscarinic acetylcholine receptor-mediated pathways in the CEM, human leukemic T-cell line.
abstract: Nitric oxide (NO) is synthesized from L-arginine by neuronal, endothelial and inducible isoforms of NO synthase (nNOS, eNOS and iNOS, respectively) and is involved in the regulation of a variety of physiological functions, including immune activity. In vascular endothelial cells, stimulation of M(3) subtype of muscarinic acetylcholine receptors (mAChRs) triggers NO synthesis by eNOS. Human lymphocytes express several mAChR subtypes and their stimulation increases the intracellular free Ca(2+) concentration and up-regulates c-fos gene expression. While the above findings suggest involvement of the lymphocytic cholinergic system in the regulation of immune function, little is known on NOS expression and NO synthesis in T-lymphocytes. In the present study, using reverse transcription-polymerase chain reaction, we found that CEM cells express mRNAs encoding iNOS and nNOS, but not for eNOS. In addition, using quantitative fluorescence microscopy and a novel NO-sensitive fluorescent indicator, DAF-2, we found that oxotremorine-M (Oxo-M) (100 microM), a non-selective mAChR agonist, enhances NO production in the cells. This effect of Oxo-M was antagonized by pirenzepine (10 microM), an antagonist acting preferentially at M(1) mAChR and by atropine (10 microM). Also 4-DAMP (10 microM), an antagonist acting preferentially at M(3) mAChR, reduced significantly the effect of Oxo-M, while AFDX-116 (10 microM), an antagonist acting preferentially at M(2) mAChR, was ineffective. These findings suggest that T-lymphocytes express functional mAChRs linked to NO synthesis by nNOS and/or iNOS.
authors.affiliation: Department of Pharmacology, Kyoritsu College of Pharmacy, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512, Japan.
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Kamimura
authors.forename: Takeshi
authors.initials: T
authors.lastname: Fujii
authors.forename: Hirotatsu
authors.initials: H
authors.lastname: Kojima
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Nagano
authors.forename: Koichiro
authors.initials: K
authors.lastname: Kawashima
mesh_terms: D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis; D015458:Leukemia, T-Cell / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000096:biosynthesis*; D011976:Receptors, Muscarinic / Q000378:metabolism*; D013601:T-Lymphocytes / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D007527:Isoenzymes; D012333:RNA, Messenger; D011976:Receptors, Muscarinic; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase