IDENTIFICATION AND MAPPING OF KERATINOCYTE MUSCARINIC ACETYLCHOLINE-RECEPTOR SUBTYPES IN HUMAN EPIDERMIS

Acetylcholine mediates cell-to-cell communications in the skin. Human epidermal keratinocytes respond to acetylcholine via two classes of ce ll-surface receptors, the nicotinic and the muscarinic cholinergic rec eptors, High affinity muscarinic acetylcholine receptors (mAChR) have been found on keratinocyte cell surfaces at high density, These recept ors mediate effects of muscarinic drugs on keratinocyte viability, pro liferation, adhesion, lateral migration, and differentiation. In this study, we investigated the molecular structure of keratinocyte mAChR a nd their location in human epidermis, Polymerase chain reaction amplif ication of cDNA sequences uniquely present within the third cytoplasmi c loop of each subtype demonstrated the expression of the mi, m3, m4, and m5 mAChR subtypes, To visualize these mAChR, we raised rabbit anti -sera to synthetic peptide analogs of the carboxyl terminal regions of each subtype, The antibodies selectively bound to keratinocyte mAChR subtypes in immunoblotting membranes and epidermis, both of which coul d be abolished by preincubating the anti-serum with the peptide used f or immunization, The immunofluorescent staining patterns produced by e ach antibody in the epidermis suggested that the profile of keratinocy te mAChR changes during epidermal turnover. The semiquantitative analy sis of fluorescence revealed that basal cells predominantly expressed m3, prickle cells had equally high levels of m4 and m5, and granular c ells mostly possessed mi, Thus, the results of this study demonstrate for the first time the presence of mi, m3, m4, and m5 mAChR in epiderm al keratinocytes. Because keratinocytes express a unique combination o f mAChR subtypes at each stage of their development in the epidermis, each receptor may regulate a specific cell function. Hence, a single c ytotransmitter, acetylcholine, and muscarinic drugs may exert differen t biologic effects on keratinocytes at different stages of their matur ation.