CRAC channels are required for [Ca2 +]i oscillations and c-fos gene expression after muscarinic acetylcholine receptor activation in leukemic T cells

Abstract

AimsT lymphocytes express muscarinic acetylcholine receptors (mAChRs) involved in regulating their proliferation, differentiation and cytokine release. Activation of M1, M3 or M5 mAChRs increases the intracellular Ca2+ concentration ([Ca2+]i) through inositol-1,4,5-phosphate (IP3)-mediated Ca2+ release from endoplasmic reticulum Ca2+ stores. In addition, T lymphocytes express Ca2+-release activated Ca2+ (CRAC) channels to induce Ca2+ influx and to regulate diverse immune functions. Our aim in the present study was to assess the role of CRAC channels during mAChR activation in the Ca2+-dependent transduction that contributes to the regulation of T cell function. Main methodsChanges in [Ca2+]i following mAChR activation on human leukemic T cells, CCRF-CEM (CEM), were monitored using fura-2, based on the ratio of 510nm fluorescences elicited by excitation at 340nm and 380nm (R340/380). Key findingsWe demonstrate that CEM cells express mainly M3 and M5 mAChRs, but little the M1 subtype, and that oxotremorine-M (Oxo-M), an mAChR agonist, induces an initial transient increase in [Ca2+]i followed by repetitive [Ca2+]i oscillations. Removing extracellular Ca2+ or pharmacological blockade of CRAC channels abolished the [Ca2+]i oscillations without affecting the initial [Ca2+]i transient induced by Oxo-M. Moreover, CRAC channel blockade also suppressed Oxo-M-induced c-fos and interleukin-2 expression. SignificanceThese results suggest that upon M3 or M5 mAChR activation, IP3-mediated Ca2+ release induces extracellular Ca2+ influx through CRAC channels, which generates repetitive [Ca2+]i oscillations and, in turn, enhances c-fos gene expression in T lymphocytes.