Excessive Accumulation of Intracellular Ca2+ After Acute Exercise Potentiated Impairment of T-cell Function

Abstract

Ca²⁺ is an important intracellular second messenger known to regulate several cellular functions. This research aimed to investigate the mechanisms of exercise-induced immunosuppression by measuring intracellular calcium levels, Ca²⁺-regulating gene expression, and agonist-evoked proliferation of murine splenic T lymphocytes. Mice were randomly assigned to the control, sedentary group (C), and three experimental groups, which performed a single bout of intensive and exhaustive treadmill exercise. Murine splenic lymphocytes were separated by density-gradient centrifugation immediately (E0), 3h (E3), and 24h after exercise (E24). Fura-2/AM was used to monitor cytoplasmic free Ca²⁺ concentration in living cells. The combined method of carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling and flow cytometry was used for the detection of T cell proliferation. The transcriptional level of Ca²⁺-regulating genes was quantified by using qPCR. Both basal intracellular Ca²⁺ levels and agonist (ConA, OKT3, or thapsigargin)-induced Ca²⁺ transients were significantly elevated at E3 group (p<0.05 vs. control). However, mitogen-induced cell proliferation was significantly decreased at E3 group (p<0.05 vs. control). In parallel, the transcriptional level of plasma membrane Ca²⁺-ATPases (PMCA), sarco/endoplasmic reticulum Ca²⁺-ATPases (SERCA), TRPC1, and P2X7 was significantly downregulated, and the transcriptional level of IP₃R2 and RyR2 was significantly upregulated in E3 (p<0.01 vs. control). In summary, this study demonstrated that acute exercise affected intracellular calcium homeostasis, most likely by enhancing transmembrane Ca²⁺ influx into cells and by reducing expression of Ca²⁺-ATPases such as PMCA and SERCA. However, altered Ca²⁺ signals were not transduced into an enhanced T cell proliferation suggesting other pathways to be responsible for the transient exercise-associated immunosuppression.

Keywords: Fura-2(AM); acute exercise; calcium channels; calcium homeostasis; cell proliferation; intracellular Ca2+.