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Background/Aims: T-lymphocyte activation and function critically depends on Ca2+ signaling, which is regulated by store operated Ca2+ entry (SOCE). Human and mouse T lymphocytes express AMP activated kinase AMPKα1, which is rapidly activated following elevation of cytosolic Ca2+ concentration ([Ca2+]i) by treatment of the cells with Ca2+ ionophore or following inhibition of endosomal Ca2+ ATPase with thapsigargin. AMPK is further activated by triggering of the T cell antigen receptor (TCR). The present study explored whether AMPK influences Ca2+ entry and Ca2+-sensitive regulation of T-lymphocyte function. Methods: T-lymphocytes were isolated and cultured from AMPKα1-deficient (ampk-/-) mice and from their wildtype (ampk+/+) littermates. The phenotype of the cells was analysed by flow cytometry, [Ca2+]i estimated from Fura-2 fluorescence, SOCE from increase of [Ca2+]i following thapsigargin treatment (1 µM), and cell function analysed by measuring cytokine secretion and western blotting. Results: Expression of surface markers in CD4+ and CD8+ T-cells were similar in ampk-/- and ampk+/+ T-lymphocyte blasts. Moreover, total STIM1 protein abundance was similar in ampk-/- and ampk+/+ T-lymphocyte blasts. However, Orai1 cell membrane protein abundance was significantly higher in ampk-/- than in ampk+/+ T-lymphocyte blasts. SOCE and increase of [Ca2+]i following TCR activation by triggering TCR with anti-CD3 and cross-linking secondary antibody were both significantly more pronounced in ampk-/- than in ampk+/+ T-lymphocyte blasts. The difference of Ca2+ entry between ampk-/- and ampk+/+ T-lymphocytes was abrogated by Orai1 inhibitor 2-aminoethoxydiphenyl borate (2-APB, 50 µM). Proliferation of unstimulated ampk-/- lymphocytes was higher than proliferation of ampk+/+ T-lymphocytes, a difference reversed by Orai1 silencing. Conclusions: AMPK downregulates Orai1 and thus SOCE in T-lymphocytes and thus participates in negative feed-back regulation of cytosolic Ca2+ activity.