ACTIVATION OF KERATINOCYTE NICOTINIC CHOLINERGIC RECEPTORS STIMULATESCALCIUM INFLUX AND ENHANCES CELL-DIFFERENTIATION

Human epidermal keratinocytes synthesize, secrete, and degrade acetylc holine and use their cell-surface nicotinic and muscarinic cholinergic receptors to mediate the autocrine and paracrine effects of acetylcho line, Because acetylcholine modulates transmembrane Ca2+ transport and intracellular metabolism in several types of cells, we hypothesized t hat cholinergic agents might have similar effects on keratinocytes, Ni cotine increased in a concentration-dependent manner the amount of Ca- 45(2+) taken up by keratinocytes isolated from human neonatal foreskin s, This effect was abolished in the presence of the specific nicotinic antagonist mecamylamine, indicating that it was mediated by keratinoc yte nicotinic acetylcholine receptor(s), The sequences encoding the al pha 5 and alpha 7 nicotinic receptor subunits were amplified from cDNA isolated from cultured keratinocytes, These subunits, as well as the alpha 3, beta 2, and beta 4 subunits previously found in keratinocytes , can be components of Ca2+-permeable nicotinic receptor channels. To learn how activation of keratinocyte nicotinic receptors affected the rate of cell differentiation, we measured the nicotinic cholinergic ef fects on the expression of differentiation markers by cultured keratin ocytes. Long-term incubations with micromolar concentrations of nicoti ne markedly increased the number of cells forming cornified envelopes and the number of cells staining with antibodies to suprabasal keratin 10, transglutaminase type I, involucrin, and filaggrin. The increased production of these differentiation-associated proteins was verified by Western blotting, Because nicotinic cholinergic stimulation causes transmembrane Ca2+ transport into keratinocytes, and because changes i n concentrations of intracellular Ca2+ are known to alter various kera tinocyte functions, including differentiation, the subcellular mechani sms mediating the autocrine and paracrine actions of epidermal acetylc holine on keratinocytes may involve Ca2+ as a second messenger.