IN INFLAMMATORY REACTIVE ASTROCYTES CO-CULTURED WITH BRAIN ENDOTHELIAL CELLS NICOTINE-EVOKED Ca2+ TRANSIENTS ARE ATTENUATED DUE TO INTERLEUKIN-1β RELEASE AND REARRANGEMENT OF ACTIN FILAMENTS

The aim of this study was to investigate whether nicotine acetylcholine receptors (nAChRs) are expressed in a more pronounced way in astrocytes co-cultured with microvascular endothelial cells from adult rat brain, compared with monocultured astrocytes, as a sign of a more developed signal transduction system. Also investigated was whether nicotine plays a role in the control of neuroinflammatory reactivity in astrocytes. Ca2+ imaging experiments were performed using cells loaded with the Ca2+ indicator Fura-2/AM. Co-cultured astrocytes responded to lower concentrations of nicotine than did monocultured astrocytes, indicating that they are more sensitive to nicotine. Co-cultured astrocytes also expressed a higher selectivity for alpha 7nAChR and alpha 4/beta 2 subunits and evoked higher Ca2+ transients compared with monocultured astrocytes. The Ca2+ transients referred to are activators of Ca2+-induced Ca2+ release from intracellular stores, both IP3 and ryanodine, triggered by influx through receptor channels. The nicotine-induced Ca2+ transients were attenuated after incubation with the inflammatory mediator lipopolysaccharide (LPS), but were not attenuated after incubation with the pain-transmitting peptides substance P and calcitonin-gene-related peptide, nor with the infection and inflammation stress mediator, leptin. Furthermore, LPS-induced release of interieukin-1 beta (IL-1 beta) measured by enzyme-linked immunosorbent assay (ELISA) was more pronounced in co-cultured versus monocultured astrocytes. Incubation with both LPS and IL-1 beta further attenuated nicotine-induced Ca2+ response. We also found that LPS and IL-1 beta induced rearrangement of the F-actin filaments, as measured with an Alexa488-conjugated phalloidin probe. The rearrangements consisted of increases in ring formations and a more dispersed appearance of the filaments. These results indicate that there is a connection between a dysfunction of nicotine Ca2+ signaling in inflammatory reactive astrocytes and upregulation of IL-1 beta and the rearrangements of actin filaments in the cells. (C) 2009 IBRO. Published by Elsevier Ltd. All rights reserved.