Lipopolysaccharide-induced down-regulation of Ca2+ release-activated Ca2+ currents (I CRAC) but not Ca2+-activated TRPM4-like currents (I CAN) in cultured mouse microglial cells.

Abstract

Microglia are the main immunocompetent cells of the mammalian central nervous system (CNS). Activation of cultured microglial cells and subsequent release of nitric oxide and cytokines critically depends on intracellular calcium levels. Since microglia undergo dramatic morphological, biochemical and electrophysiological changes in response to pathological events in the CNS, we investigated temporal changes in expression levels of ion channels involved in cellular calcium homeostasis in mouse cortical microglial cells in culture. Specifically, we assessed the inward and delayed outward rectifier potassium currents (I IRK and I DRK), calcium (Ca2+) release-activated Ca2+ currents (I CRAC) and Ca2+-activated TRPM4-like currents (I CAN) in non-activated microglia and cells that were activated by exposure to lipopolysaccharide (LPS) between 3 and 48 h. Unstimulated microglial cells, subcultured from an astrocyte coculture, typically exhibited a ramified, rod-shaped morphology. During the first 3 days of culture cell size and shape were maintained, but the percentage of cells showing prominent I IRK went up and those expressing I DRK went down. Cells retaining I DRK exhibited smaller amplitudes, whereas those of I IRK and I CRAC were not affected. However, after 24 h of exposure to 1 microg ml(-1) LPS, most cells showed an amoeboid ('fried egg'-shaped) morphology with a 62% increase in cell capacitance. At that point in time, only 14% of the cells revealed I IRK and 3% had I DRK exclusively, whereas the majority of cells expressed both currents. The amplitudes of I CRAC and I IRK progressively decreased after stimulation, whereas I DRK transiently reached a maximum after 6 h of LPS exposure and then returned to pre-stimulation expression levels. Cultured microglia also revealed TRPM4-like, Ca2+-activated non-selective currents (I CAN) with an EC50 of 1.2 microm [Ca2+]i. The expression levels of this current did not change significantly during and after 24 h of LPS exposure. We propose that LPS-induced down-regulation of I IRK and I CRAC will reduce the cell's capacity to produce significant calcium influx upon receptor activation and result in decreased sensitivity to exogenous stimulation. In this scenario, I CAN expression would remain constant, although its activity would automatically be reduced due to the diminished calcium influx capacity of the cell.

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