Acute actions of tumor necrosis factor-alpha on intracellular Ca(2+) and K(+) currents in human microglia.

The effects of acute application of the pro-inflammatory cytokine tumor necrosis factor-alpha (TNFalpha) on levels of intracellular Ca(2+) ([Ca(2+)]i) and on whole-cell outward and inward K(+) currents were studied in cultured human microglia. TNFalpha elicited a linear increase in [Ca(2+)]i to a plateau level in microglia bathed in either standard physiological saline solution or Ca(2+)-free physiological saline solution. The rate of increase of [Ca(2+)]i or the level of [Ca(2+)]i attained was not significantly altered in the absence of external Ca(2+) indicating that Ca(2+) influx did not contribute appreciably to the cytokine-induced rise in [Ca(2+)]i. This point was directly confirmed using Mn(2+) quenching where no change in signal fluorescence was observed with TNFalpha treatment of microglia in Ca(2+)-free physiological saline solution. The rate of increase of [Ca(2+)]i induced by TNFalpha in Ca(2+)-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 TNFalpha 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 TNFalpha had no effect to alter inward rectifier currents generated from voltage ramps. The signaling pathways involving TNFalpha modulation of [Ca(2+)]i and K(+) channels in human microglia may contribute to functional and pathological actions of the cytokine in the brain.

Anion channels modulate store-operated calcium influx in human microglia.

Recent work from this laboratory has demonstrated that purinergic-mediated depolarization of human microglia inhibited a store-operated pathway for entry of Ca2+. We have used Fura-2 spectrofluorometry to investigate the effects on store-operated Ca2+ influx induced by replacement of NaCl with Na-gluconate in extracellular solutions. Three separate procedures were used to activate store-operated channels. Platelet activating factor (PAF) was used to generate a sustained influx of Ca2+ in standard physiological saline solution (PSS). The magnitude of this response was depressed by 70% after replacement of PSS with low Cl- PSS. A second procedure used ATP, initially applied in Ca2+-free PSS solution to deplete intracellular stores. The subsequent perfusion of PSS solution containing Ca2+ resulted in a large and sustained entry of Ca2+, which was inhibited by 75% with low Cl- PSS. The SERCA inhibitor cyclopiazonic acid (CPA) was used to directly deplete stores in zero-Ca2+ PSS. Following the introduction of PSS containing Ca2+, a maintained stores-operated influx of Ca2+ was evident which was inhibited by 77% in the presence of the low Cl- PSS. Ca2+ influx was linearly reduced with cell depolarization in elevated K+ (7.5 to 35 mM) suggesting that changes in external Cl- were manifest as altered electrical driving force for Ca2+ entry. However, 50 mM external KCl effectively eliminated divalent entry which may indicate inactivation of this pathway with high magnitudes of depolarization. Patch clamp studies showed low Cl-PSS to cause depolarizing shifts in both holding currents and reversal potentials of currents activated with voltage ramps. The results demonstrate that Cl- channels play an important role in regulating store-operated entry of Ca2+ in human microglia.

Acute application of interleukin-1beta induces Ca(2+) responses in human microglia.

The effects of the pro-inflammatory cytokine interleukin-1-beta (IL-1beta) on levels of intracellular calcium [Ca(2+)](i) in cultured human microglia have been studied using the fluorescent Ca(2+) indicator fura-2. IL-1beta (2 ng/ml) caused a slow, progressive increase in [Ca(2+)](i) in standard Ca(2+)-containing physiological solution (PSS). A similar effect was observed in separate studies using Ca(2+)-free PSS, however, the mean rate of increase was significantly lower than that measured with PSS. Similar results were obtained in a separate protocol, where cells were exposed to both IL-1beta in Ca(2+)-free PSS and PSS. The slope of the IL-1beta induced increase of [Ca(2+)](i) in Ca(2+)-free PSS was not altered when adenosine triphosphate was added prior to application of the cytokine. These results suggest that IL-1beta-induced responses in human microglia involve both a Ca(2+) entry pathway and a mechanism of intracellular increase other than from IP(3)-sensitive stores.