Fluorescence measurement of changes in intracellular calcium induced by excitatory amino acids in cultured cortical astrocytes

Abstract

Population response of [Ca2+]i in cultured cortical astrocytes to excitatory amino acids was measured at room temperature using the calcium-sensitive dye fura-2. Quisqualic acid (QA), glutamate (Glu), and kainic acid (KA) caused a peak increase in [Ca2+]i in the order QA greater than Glu greater than KA. No response to N-methyl-D-aspartic acid (NMDA) was observed whether or not Mg2+ was present externally. Both QA and Glu (100 microM) frequently elicited a decaying oscillatory [Ca2+]i response during sustained agonist application; the period of oscillations initially was 23.5 sec and increased as the response was damped. Comparatively, the [Ca2+]i response to KA was nonoscillatory. Both responses to Glu and KA were reduced slightly by antagonist gamma- D-glutamylaminomethyl-sulfonic acid (1 mM), but virtually were abolished by kynurenic acid (3 mM). Replacement of external Na+ by choline had no significant effect on the Glu response. Removal of external Ca2+ reduced the peak response to QA, Glu, and KA to 40, 34, and 18%, respectively; and markedly reduced the degree of QA- and Glu- induced [Ca2+]i oscillations. Pretreatment with phorbol esters, a potent activator of protein kinase C, blocked the [Ca2+]i response to Glu but not KA. It is concluded that cortical astrocytes express Glu receptors of the non-NMDA type in culture and that receptor activation leads to Ca2+ influx and release of internal Ca2+. Mobilization of Ca2+ apparently occurs via the known Glu-mediated hydrolysis of inositol lipids, which may come under negative-feed-back control by protein kinase C activation. Oscillatory [Ca2+]i signaling offers the possibility of a dynamic population response in an electrically coupled glial network.

Diversity of Calcium Signaling by Metabotropic Glutamate Receptors

“…Similar to the observations in mGluR5a-expressed HEK-293 cells (9), the PKC activator abolishes Ca 2ϩ response in cultured astrocytes (21,23). Moreover, both PKC inhibitor and PP1/PP2A phosphatase inhibitor convert Ca 2ϩ response from an oscillatory to non-oscillatory pattern, suggesting that the same mechanism underlies the generation of [Ca 2ϩ ] i oscillations in mGluR5-expressed heterologous and native cells (21).…”

The Metabotropic Glutamate Receptor mGluR5 Induces Calcium Oscillations in Cultured Astrocytes via Protein Kinase C Phosphorylation

“…1D). Cells cultured in the presence of FCS showed a flat shape, thus representing mostly type-l astrocytes (Jensen and Chiu, 1990) (data not shown), but these cells attained a branched, stellate shape when FCS was removed from the culture medium (Fig. lE).…”

“…After 6 days, the flask was shaken mechanically at 280 rpm overnight on a horizontal orbital shaker (New Brunswick Scientific Co., Edison, NJ, U.S.A.), and the top layer of cells was removed. This procedure removed most of the oligodendrocytes, microglia, and processbearing type-2 astrocytes and thus yielded mainly type-1 astrocytes with a flat shape (Jensen and Chiu, 1990). One day after this purification step, secondary astrocyte cultures were established by trypsinizing the primary culture and subplating onto poly-o-lysine-precoated plastic dishes in DMEM p!us 10% FCS.…”

Regulation of neuregulin expression in the injured rat brain and cultured astrocytes

“…C ells were maintained in DM EM containing 10% FC S for 6 d. Next, the flasks were shaken mechanically at 200 rpm overnight in a horizontal orbital shaker to remove the top layer of cells. This procedure removed most of the oligodendrocytes, microglia, and type 2 astrocytes and thus yielded mainly type-1 astrocytes with a flat shape (Jensen and Chiu, 1990). One day after this purification step, secondary astrocyte cultures were established by trypsinizing the primary culture and subplating it onto poly-Dlysine-precoated plastic dishes in DM EM supplemented with 10% FC S.…”