Ca²⁺ oscillation and c-fos gene expression induced via muscarinic acetylcholine receptor in human T- and B-cell lines

Abstract.

We previously reported that blood acetylcholine (ACh) mainly originates from T-lymphocytes and that muscarinic (Ms) ACh receptor mRNA is expressed in both T- and B-lymphocytes. In the present study, we used confocal laser scanning microscopy and fluo-3, a calcium-sensitive indicator, to investigate the effects of Ms-ACh receptor agonists on the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in single cells from human T-cell (CEM) and B-cell (Daudi) lines, which we used as models of lymphocytes. In both cell lines, stimulation of Ms-ACh receptors with ACh (0.1–100 µM), bethanechol (100 µM), carbachol (100 µM) or oxotremorine-M (Oxo-M; 0.1–100 µM) induced [Ca²⁺]_i-dependent increases in fluo-3 fluorescence, which in the presence of extracellular Ca²⁺ were followed by oscillations in [Ca²⁺]_i that persisted for at least 10 min. All effects were completely blocked by atropine (1 µM), an Ms-ACh receptor antagonist. In both cell lines Oxo-M (100 µM) up-regulated expression of c-fos mRNA in an extracellular Ca²⁺-dependent manner. Again, the effect was blocked by 1 µM atropine. These results provide the first evidence that stimulation of Ms-ACh receptors induces Ca²⁺ oscillations and up-regulates c-fos gene expression in T- and B-lymphocytes, which is consistent with the notion that ACh released from T-lymphocytes triggers nuclear signaling via Ms-ACh receptors.