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Regulation of intracellular free calcium and electrophysiological properties of cultured human microglia

University of British Columbia

This study was performed to investigate the changes in the concentration of free calcium ([Ca²+]i) in cultured human microglia in response to ATP and cholinergic agonists by using calcium microfluorescence imaging measurements with the fluorescent dye fura-2. Whole-cell patch-clamp recording was also used to study the effects of ATP on the properties of membrane currents of human microglia. Microglia are the representatives of macrophages in the brain, and serve specific functions in the defense of the central nerves system (CNS) against microorganisms. An important function of microglia in the CNS is a rapid response to neuronal damage, thus, they can be mobilized rapidly to deal with different pathological conditions. The application of ATP (100 μM) to microglia led to a transient [Ca²+]i increase with two components, an initial increase and a delayed plateau. The initial increase was also observed in Ca²+-free media suggesting it is due to the release of Ca²+ from internal stores. The elevated [Ca²+]i plateau is due to Ca²+ influx. The application of either 100 μM carbachol (CCh), or 100 μM acetylcholine (ACh) to microglia elicited a transient [Ca²+]i increase which was also composed of two phases, an initial spike and a sustained plateau. This initial spike also appeared in the Ca²+-free recording buffer. The CCh-induced [Ca²+]i increase was atropine-sensitive, and was largely mediated by release of Ca²+ from internal stores, followed by Ca²+ influx. Both the ATP- and CCh-produced [Ca²+]i increases in microglia desensitized to repeated agonist exposures. ATP (100 μM) produced a transient activation of an inward non-selective cation current at a holding potential of -60 mV. The ATP responses in whole-cell recording also included an enhanced transient expression of an outward K+ current activated with applied depolarizing steps and an increase in inward K+ conductance. When ATP or CCh exposures were performed in the absence of external Ca²+, adding back external Ca²+ always produced a large [Ca²+]i increase. Similar observations were made when the store depleting membrane ATP-ase inhibitor thapsigargin (Tg) was applied to the cell in Ca²+-free conditions or by incubating the cell in Ca²+-free buffer for a few minutes. This external Ca²+ addition induced a [Ca²+]i increase suggesting that capacitative Ca²+ entry mechanisms exist in human microglia. Our results demonstrate [Ca²+]i responds to extracellular ATP and cholinergic agonists. We also find expression of depolarizing inward cation currents and outward K+ currents to ATP which are not present in control condition. These data suggest that ATP and cholinergic agonists could act as signals to stimulate the transition of microglia from a resting state to an activated state under some circumstances in the CNS, and could be involved in the pathology of CNS disease. [Scientific formulae used in this abstract could not be reproduced.]

Thesis/Dissertation

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2009-05-28

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http://hdl.handle.net/2429/8382

Neuroscience

University of British Columbia

UBCV

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