KERATINOCYTE MUSCARINIC ACETYLCHOLINE-RECEPTORS - IMMUNOLOCALIZATION AND PARTIAL CHARACTERIZATION

We have reported previously that human keratinocytes synthesize and se crete acetylcholine and that muscarinic cholinergic drugs have effects on keratinocyte proliferation, adhesion, and migration. This study de fines the location of muscarinic acetylcholine receptors in human epid ermis and describes some pharmacologic and molecular properties of the se receptors, Confocal microscopy employing the anti-muscarinic recept or monoclonal antibody M35 visualized the receptors in the intercellul ar areas of normal human epidermis. Using immunoelectron microscopy, t he receptors appeared to be attached to the keratinocyte plasma membra nes. Functional, high-density (Bmax = 8.3 nmol/2 x 10(6) cells) and hi gh-affinity (Kd = 21.5 nM) muscarinic receptors were demonstrated by s aturable binding of the reversible radioligand [H-3]quinuclidinyl benz ilate to the surfaces of freshly isolated epidermal cells at 0 degrees C. Receptor proteins were separated by gel electrophoresis. An appare nt isoelectric point of pH 4.3 was determined in immunoblots of sodium -cholate-solubilized receptors separated on isoelectric-focusing gels. Three protein bands, two at approximately 60 kDa and one at 95 kDa, w ere visualized in immunoblots of membrane-bound or solubilized recepto rs separated by sodium dodecylsulfate-polyacrylamide gel electrophores is. The covalent, irreversible ligand [H-3]propylbenzilylcholine musta rd confirmed these results. Thus, human keratinocytes express a hetero geneous population of muscarinic cholinergic receptors. Because human keratinocytes also express nicotinic cholinergic receptors, endogenous ly secreted acetylcholine may control different biologic processes in these cells by activating different types of their cholinergic recepto rs.