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Nitric oxide (NO) synthase mRNA expression and NO production via muscarinic acetylcholine receptor-mediated pathways in the CEM, human leukemic T-cell line.

机译:一氧化氮(NO)合酶mRNA表达和通过毒蕈碱乙酰胆碱受体介导的途径在CEM人类白血病T细胞系中产生。

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摘要

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.
机译:一氧化氮(NO)是由L-精氨酸通过一氧化氮合酶的神经元,内皮和诱导型亚型(分别为nNOS,eNOS和iNOS)合成的,并参与多种生理功能的调节,包括免疫活性。在血管内皮细胞中,毒蕈碱性乙酰胆碱受体(mAChRs)的M(3)亚型的刺激触发eNOS的NO合成。人类淋巴细胞表达几种mAChR亚型,它们的刺激增加细胞内游离Ca(2+)的浓度并上调c-fos基因表达。尽管以上发现表明淋巴细胞胆碱能系统参与了免疫功能的调节,但对于T淋巴细胞中NOS的表达和NO的合成知之甚少。在本研究中,使用逆转录聚合酶链反应,我们发现CEM细胞表达编码iNOS和nNOS的mRNA,但不表达eNOS。此外,使用定量荧光显微镜和新型NO敏感荧光指示剂DAF-2,我们发现非选择性mAChR激动剂oxotremorine-M(Oxo-M)(100 microM)增强了细胞中NO的产生。 Oxo-M的这种作用被吡仑西平(10 microM)(优先作用于M(1)mAChR的拮抗剂)和阿托品(10 microM)拮抗。同样优先作用于M(3)mAChR的拮抗剂4-DAMP(10 microM)显着降低了Oxo-M的作用,而优先作用于M(2)mAChR的拮抗剂AFDX-116(10 microM)被降低了。无效的。这些发现表明,T淋巴细胞表达功能性mAChR,其通过nNOS和/或iNOS与NO合成相关。

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