LOCALIZATION OF AMINOPEPTIDASE ACTIVITY IN FRESHLY EXCISED HUMAN SKIN- DIRECT VISUALIZATION BY CONFOCAL LASER-SCANNING MICROSCOPY

The aim of this study was to localize and visualize aminopeptidase act ivity within freshly excised, dermatomed human skin without perturbati on of its histologic integrity. The use of confocal laser scanning mic roscopy (CLSM) is introduced as a novel approach by which to monitor t he degradation of suitable substrates in the skin. The fluorescence of the metabolites originating from the cleavage of the aminopeptidase p robe bis-Leu-rhodamine 110 (Leu(2)-R110) was interpreted to reflect th e local aminopeptidase activity in the tissue, To separate the kinetic s of diffusion and degradation of Leu(2)-R110, a lateral application m ode was introduced: the probe was applied at the cutting plane of a me chanical cross-section of the sample, and optical cross-sections were made parallel to the cutting plane of the mechanical section. By this means, simultaneous and equal access of the substrate to the various s trata and domains of the skin was achieved, The observations revealed that the fluorescence, i.e., aminopeptidase activity, was evenly distr ibuted throughout the viable part of the epidermis, with enhanced fluo rescence (''hot spots'') in the upper layers of the stratum granulosum , while dermis and stratum corneum showed considerably less aminopepti dase activity. Independent studies with hair follicles (obtained from trypsin-separated stratum corneum) also showed aminopeptidase activity , mostly at the root sheath. Because of the advantage of direct visual ization and localization of enzymatic activity in intact tissue, the l ateral application mode of substrate administration in combination wit h CLSM may be beneficial to further elucidate the location and intensi ty of metabolic activity in other living tissues as well.