IL-2-induced NF-κB phosphorylation upregulates cation nonselective conductance in human cardiac fibroblasts

Abstract

Purpose: Studies of negative ionotropic effects of IL-2 create the basis for possible IL-2 impact on nonselective conductance (G_NS), which potentially makes these effects useful in elucidation of the pathways affected by IL-2.

Materials and methods: A culture of human cardiac fibroblasts (CHCFs) was used in this study. A voltage clamp mode of the whole-cell patch-clamp technique was introduced. The level of phosphorylated NF-κB was determined by newly developed semi-quantitative ELISA.

Results: The IL-2 (5 ng/ml) increased the currents during the depolarizing clamp to larger amplitudes without changing their time course. In the CHCFs pretreated with 50 μmol/L 2-APB, IL-2-induced increase in G_NS was highly prevented (p < 0.001), indicating possible STIM-ORAI involvement. The CHCF perfusion with IL-2 in the presence of IMD-0354 for 14-16 min confirmed a significant G_NS prevention (between 50 and 80%), indicating IκB involvement in the IL-2-induced signaling. The CHCF perfusion with IL-2 in the presence of Chel, induced significant prevention in the G_NS expression (between 50 and 80%) compared to IL-2 treated cells, indicating PKC involvement.

Conclusions: IL-2 mediated G_NS increase is mediated by activation of downstream players such as PKC, IκB, and NF-κB, which are probably further responsible for the upregulation of STIM-ORAI.

Keywords: Cation nonselective conductance; Human cardiac fibroblasts; IL-2; NF-κB and IκB; PKC.