In inflammatory reactive astrocytes co-cultured with brain endothelial cells nicotine-evoked Ca(2+) transients are attenuated due to interleukin-1beta 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. Ca(2+) imaging experiments were performed using cells loaded with the Ca(2+) 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 alpha7nAChR and alpha4/beta2 subunits and evoked higher Ca(2+) transients compared with monocultured astrocytes. The Ca(2+) transients referred to are activators of Ca(2+)-induced Ca(2+) release from intracellular stores, both IP(3) and ryanodine, triggered by influx through receptor channels. The nicotine-induced Ca(2+) 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 interleukin-1beta (IL-1beta) measured by enzyme-linked immunosorbent assay (ELISA) was more pronounced in co-cultured versus monocultured astrocytes. Incubation with both LPS and IL-1beta further attenuated nicotine-induced Ca(2+) response. We also found that LPS and IL-1beta 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 Ca(2+) signaling in inflammatory reactive astrocytes and upregulation of IL-1beta and the rearrangements of actin filaments in the cells.
Delbro
D
D
School of Pure and Applied Natural Sciences, University of Kalmar, Kalmar, Sweden.
Westerlund
A
A
Björklund
U
U
Hansson
E
E
D008841:Actin Cytoskeleton / Q000502:physiology*; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000187:drug effects* / Q000502:physiology; D001921:Brain / Q000166:cytology*; D015740:Calcitonin Gene-Related Peptide / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D018920:Coculture Techniques; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D053583:Interleukin-1beta / Q000378:metabolism*; D020738:Leptin / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D009538:Nicotine / Q000494:pharmacology*; D018722:Nicotinic Agonists / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013373:Substance P / Q000494:pharmacology; D013997:Time Factors
D053583:Interleukin-1beta; D020738:Leptin; D008070:Lipopolysaccharides; D018722:Nicotinic Agonists; D013373:Substance P; D009538:Nicotine; D015740:Calcitonin Gene-Related Peptide; D002118:Calcium