AGAROSE-GEL KERATINOCYTE OUTGROWTH SYSTEM AS A MODEL OF SKIN REEPITHILIALIZATION - REQUIREMENT OF ENDOGENOUS ACETYLCHOLINE FOR OUTGROWTH INITIATION

To better understand the mechanisms of skin re-epithelization, we deve loped a simple technique that assays the outgrowth of human keratinocy tes. Second-passage foreskin keratinocytes were inoculated at high cel l density into 3-mm wells cut from agarose gels in standard 6-well tis sue culture dishes. The cells settled on the dish bottom and formed a confluent colony. The cells at the periphery of the colony flattened, spread their cytoplasm, and moved away over the dish surface under the agarose gel. The morphology of migrating keratinocytes was observed m icroscopically through the transparent agarose, and the migration dist ance was measured after the gels were removed and after cells were fix ed and stained. To determine which cell activities were involved in th e outgrowth, the effects of cholinergic compounds on keratinocyte outg rowth were compared with their effects on keratinocyte proliferation, cell-plastic attachment, and spreading measured in separate sets of ex periments. Outgrowth was inhibited by the specific inhibitor of acetyl choline synthesis bromoacetylcholine (0.05 mM) and restored by 5 mM ex ogenous acetylcholine. The irreversible muscarinic antagonist propylbe nzilylcholine mustard (0.05 mM) abolished the restorative effects of e xogenous acetylcholine, and also inhibited outgrowth of intact keratin ocytes. In keratinocyte cell cultures, bromoacetylcholine stopped cell division. Propylbenzilylcholine mustard increased cell number, but in terfered with cell-plastic attachment and spreading. This suggests tha t cell-matrix attachment, spreading, and locomotion of human keratinoc ytes, but not mitosis, mediate the earliest stages of skin re-epitheli zation, and that endogenous acetylcholine regulates these keratinocyte functions. Specifically, keratinocyte acetylcholine is required to in itiate outgrowth.