Abstract

Early stages of wound healing rely on the ability of keratinocytes (KCs) to move over the denuded dermis to re-epithelialize the defect. The agarose gel keratinocyte outgrowth system (AGKOS) is an in vitro model of skin re-epithelialization designed to study the migratory function of KCs. Endogenously secreted acetylcholine controls crawling locomotion of KCs in AGKOS by binding to the cholinergic receptors of both the nicotinic and the muscarinic classes that are expressed by KCs. In this study, we used AGKOS to elucidate the nicotinic pathway of cholinergic control of keratinocyte migration. Activation of the nicotinic acetylcholine receptors decreased the migration distance of KC in a dose-dependent fashion without altering cell viability. Nicotine also increased in a dose-dependent manner transmembrane influx of ⁴⁵Ca²⁺, and caused a transient rise in the concentration of [Ca²⁺]_i. Perfect correlation between concentration responses found in the migration and⁴⁵Ca²⁺ influx assays suggested that nicotine-induced inhibition of crawling locomotion relies on modulation of Ca²⁺ metabolism in KCs. The effects of nicotine could be mediated by the α3- and the α7-containing nicotinic receptors visualized on KCs by immunostaining. Long-term incubation with nicotine up-regulated α7 and down-regulated α3 expression. Thus, nicotine exerts inhibitory effects on keratinocyte migration, and Ca²⁺ serves as a second messenger in the signaling pathway. These results help explain deleterious effects of nicotine on wound re-epithelialization, and suggest that smoking may delay wound healing via nicotinic receptor-mediated pathway.