NUCLEAR MUSCARINIC ACETYLCHOLINE-RECEPTORS IN CORNEAL CELLS FROM RABBIT

Purpose. Previous studies have indicated that muscarinic acetylcholine receptors (mAChR) may be present in an unexpected, unique location an d play a singular role in cellular growth regulation of rabbit corneal epithelium that may be of general physiologic significance if found i n other cells. The purpose of this study was to examine rabbit corneas and corneal cells in culture to determine mAChR location and tissue d istribution. Methods. Using [H-3]-propylbenzilylcholine mustard ([H-3] PrBChM), which binds covalently to the active site of mAChR, rabbit co rneal cross-sections, cultured corneal keratocytes, epithelial and end othelial cells, as well as nuclei isolated from these cultured corneal cells were labeled, stained, and autoradiographed. Nuclei labeled wit h [H-3]PrBChM were further analyzed by sodium dodecyl sulfate polyacry lamide gel electrophoresis. Results. Direct visual confirmation of the localization of mAChRs was obtained. MAChR were found in epithelial a nd endothelial layers of fresh-frozen corneal cross-sections, in cultu red rabbit epithelial and endothelial cells, and on isolated rabbit ep ithelial and endothelial cell nuclei. mAChR were not detectable in ker atocytes with these techniques. When [H-3]PrBChM-labeled nuclei from c ultured corneal cells were analyzed by sodium dodecyl sulfate polyacry lamide gel electrophoresis, epithelial and endothelial samples showed specific mAChR binding, whereas binding to keratocyte nuclei was not d etectable. Conclusions. As a result of these findings, a revised hypot hesis is suggested for the locations and possible functions of mAChR i n regulation of growth in corneal and other cells.