Activation of keratinocyte nicotinic cholinergic receptors stimulates calcium influx and enhances cell differentiation

Human epidermal keratinocytes synthesize, secrete, and degrade acetylcholine and use their cell-surface nicotinic and muscarinic cholinergic receptors to mediate the autocrine and paracrine effects of acetylcholine. Because acetylcholine modulates transmembrane Ca²⁺ transport and intracellular metabolism in several types of cells, we hypothesized that cholinergic agents might have similar effects on keratinocytes. Nicotine increased in a concentration-dependent manner the amount of ⁴⁵Ca²⁺ taken up by keratinocytes isolated from human neonatal foreskins. This effect was abolished in the presence of the specific nicotinic antagonist mecamylamine, indicating that it was mediated by keratinocyte nicotinic acetylcholine receptor(s). The sequences encoding the α5 and α7 nicotinic receptor subunits were amplified from cDNA isolated from cultured keratinocytes. These subunits, as well as the α3, β2, and β4 subunits previously found in keratinocytes, can be components of Ca²⁺-permeable nicotinic receptor channels. To learn how activation of keratinocyte nicotinic receptors affected the race of cell differentiation, we measured the nicotinic cholinergic effects on the expression of differentiation markers by cultured keratinocytes. Long-term incubations with micromolar concentrations of nicotine 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 in concentrations of intracellular Ca²⁺ are known to alter various keratinocyte functions, including differentiation, the subcellular mechanisms mediating the autocrine and paracrine actions of epidermal acetylcholine on keratinocytes map involve Ca²⁺ as a second messenger.