The Acetylcholine Signaling Network of Corneal Epithelium and Its Role in Regulation of Random and Directional Migration of Corneal Epithelial Cells

Purpose: Because cholinergic drugs are used in ophthalmology and cholinergic stimulation has been shown to facilitate epithelialization of mucocutaneous wounds, we performed a systematic analysis of components of the cholinergic network of human and murine corneal epithelial cells (CECs) and determined the role of autocrine and paracrine acetylcholine (ACh) in regulation of CEC motility.

Methods: We investigated the expression of ACh receptors at the mRNA and protein levels in human immortalized CECs, localization of cholinergic molecules in normal and wounded murine cornea, and the effects of cholinergic drugs on CEC directional and random migration in vitro, intercellular adhesion, and expression of integrin αV and E-cadherin.

Results: We demonstrated that corneal epithelium expresses the ACh-synthesizing enzyme choline acetyltransferase, the ACh-degrading enzyme acetylcholinesterase, two muscarinic ACh receptors (mAChRs), M3 and M4, and several nicotinic ACh receptors (nAChRs), including both α7- and α9-made homomeric nAChRs and predominantly the α3β2±α5 subtype of heteromeric nAChRs. Wounding affected the expression patterns of cholinergic molecules in the murine corneal epithelium. Constant stimulation of CECs through both muscarinic and nicotinic signaling pathways was essential for CEC survival and both directional and random migration in vitro. Both α7 and non-α7 nAChRs elicited chemotaxis, with the α7 signaling exhibiting a stronger chemotactic effect. Cholinergic stimulation of CECs upregulated expression of the integrin and cadherin molecules involved in epithelialization. We found synergy between the proepithelialization signals elicited by different ACh receptors expressed in CECs.

Conclusions: Simultaneous stimulation of mAChRs and nAChRs by ACh may be required to synchronize and balance ionic and metabolic events in a single cell. Localization of these cholinergic enzymes and receptors in murine cornea indicated that the concentration of endogenous ACh and the mode of its signaling differ among corneal epithelial layers. Elucidation of the signaling events elicited upon agonist binding to corneal mAChRs and nAChRs will be crucial for understanding the mechanisms of ACh signaling in CECs, which has salient clinical implications.