Acute actions of tumor necrosis factor-α on intracellular Ca2+ and K+ currents in human microglia

Abstract

The effects of acute application of the pro-inflammatory cytokine tumor necrosis factor-α (TNFα) on levels of intracellular Ca2+ ([Ca2+]i) and on whole-cell outward and inward K+ currents were studied in cultured human microglia. TNFα elicited a linear increase in [Ca2+]i to a plateau level in microglia bathed in either standard physiological saline solution or Ca2+-free physiological saline solution. The rate of increase of [Ca2+]i or the level of [Ca2+]i attained was not significantly altered in the absence of external Ca2+ indicating that Ca2+ influx did not contribute appreciably to the cytokine-induced rise in [Ca2+]i. This point was directly confirmed using Mn2+ quenching where no change in signal fluorescence was observed with TNFα treatment of microglia in Ca2+-free physiological saline solution. The rate of increase of [Ca2+]i induced by TNFα in Ca2+-free physiological saline solution was not altered by prior application of ATP to deplete inositol triphosphate stores indicating that these stores did not contribute to the cytokine response. In whole-cell patch clamp recordings, the acute treatment of human microglia with TNFα led to the expression of an outward K+ current in one-third (14 of 41) of cells. This current was activated at potentials positive to −30 mV, showed rapid kinetics of activation with no evident inactivation and had an I–V relation exhibiting outward rectification. Analysis of tail currents showed reversal of the outward K+ current near −70 mV and tetraethylammonium (10 mM) inhibited the outward K+ current to 24% of control level. Acute application of TNFα had no effect to alter inward rectifier currents generated from voltage ramps.

The signaling pathways involving TNFα modulation of [Ca2+]i and K+ channels in human microglia may contribute to functional and pathological actions of the cytokine in the brain.

Keywords human microglia; tumor necrosis factor-α; fura-2 spectrofluorometry; intracellular Ca2+; whole-cell patch clamp; K+ current