Inflammatory activation enhances NMDA-triggered Ca2+ signalling and IL-1β secretion in primary cultures of rat astrocytes

The aim of the present study was to measure the effects of NMDA receptor antagonists on rat astroglial-enriched primary cultures after incubation with lipopolysaccharide (LPS), with a view to explaining the role of NMDA receptors in the inflammatory activation of astrocytes. First, the presence of NMDA receptor subunits was confirmed at the protein level by immunocytochemical methods. The presence of functional NMDA receptors containing GluN2B subunits was then established by ratiometric fluorescent Ca(2+) imaging which revealed transient NMDA-triggered Ca(2+) responses. These responses could be blocked by the competitive antagonist 2-amino-5-phosphonopentoate (APV) and the non-competitive GluN2B subunit-selective antagonist ifenprodil. The NMDA-evoked Ca(2+) transients were dependent on Ca(2+) release from intracellular stores via interaction with InsP3-sensitive receptors as they were blocked by thapsigargin or xestospongin C. Following 24h incubation with LPS, astroglial inflammatory activation increased IL-1β secretion and NMDA-triggered Ca(2+) transients. The addition of APV or ifenprodil inhibited these enhanced responses, suggesting that LPS exposure stimulates IL-1β release from astrocytes through a mechanism that requires NMDA receptor stimulation.