Receptor-mediated inhibition of keratinocyte migration by nicotine involves modulations of calcium influx and intracellular concentration

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 g el keratinocyte outgrowth system (AGKOS) is an in vitro model of skin re-ep ithelialization designed to study the migratory function of KCs. Endogenous ly secreted acetylcholine controls crawling locomotion of KCs in AGKOS by b inding to the cholinergic receptors of both the nicotinic and the muscarini c classes that are expressed by KCs. In this study, we used AGKOS to elucid ate the nicotinic pathway of cholinergic control of keratinocyte migration. Activation of the nicotinic acetylcholine receptors decreased the migratio n distance of KC in a dose-dependent fashion without altering cell viabilit y. Nicotine also increased in a dose-dependent manner transmembrane influx of Ca-45(2+) and caused a transient rise in the concentration of [Ca2+](i). Perfect correlation between concentration responses found in the migration and Ca-45(2+) influx assays suggested that nicotine-induced inhibition of crawling locomotion relies on modulation of Ca2+ metabolism in KCs. The eff ects of nicotine could be mediated by the alpha 3- and the alpha 7-containi ng nicotinic receptors visualized on KCs by immunostaining. Long-term incub ation with nicotine upregulated alpha 7 and down-regulated alpha 3 expressi on. 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-me diated pathway.