TRANSPORT AND HYDROLYSIS OF ENKEPHALINS IN CULTURED ALVEOLAR EPITHELIAL MONOLAYERS

An in vitro cultured monolayer system of alveolar epithelial cells was used as a model to investigate transport and hydrolysis of two enkeph alin peptides, Met-enkephalin (TGGPM) and [D-Ala2]Met-enkephalinamide (TAGPM), in pulmonary epithelium. Isolated alveolar type II cells form ed continuous monolayers when grown on microporous tissue culture-trea ted polycarbonate filters in serum-free, hormonally defined medium. Tr ansport and hydrolysis studies of enkephalins in the monolayer system obtained after 6 days in culture, using fluorescence reversed-phase HP LC, indicate a reduced but significant degradation of enkephalins in t he alveolar epithelium compared to most other epithelia previously rep orted. Aminopeptidases and dipeptidyl carboxypeptidase represent two m ajor hydrolytic enzymes for TGGPM, as indicated by the formation of th e degradative products Tyr and Tyr-Gly-Gly, while dipeptidyl peptidase , which is responsible for the formation of Tyr-Gly, contributes much less. The enkephalinase inhibitor thiorphan failed to prevent the hydr olysis of TGGPM whereas the enkephalin analog TAGPM was relatively res istant to enzymatic cleavage. The rate of enkephalin transport across the alveolar epithelium was directly proportional to drug concentratio n and occurred irrespective of transport direction, suggesting passive diffusion as the major mechanism for transepithelial transport. Agent s that affect paracellular transport pathways, e.g.. EGTA and the calc ium ionophore A-23187, greatly promoted the transport rate. The ionoph ore at high doses, in addition to promoting tight junction permeabilit y, also caused cellular damage associated with a sustained rise in int racellular calcium levels, as indicated by nuclear propidium iodide fl uorescence. The cultured monolayer of alveolar epithelium may be used to study pulmonary drug absorption, degradation, and toxicity.