Characterization of proteolytic activities of pulmonary alveolar epithelium

Abstract

Pulmonary alveolar type I epithelial cell and its progenitor, type II cell, present major transport and enzyme barriers for systemic delivery of pulmonary administered peptide drugs. The present study investigates the effect of cellular differentiation of type II to type I cells on their proteolytic activities, and evaluates the suitability of a continuous lung cell line, A549, for drug transport and degradation studies. High performance liquid chromatography was used to assess the degradation kinetics of two model peptide substrates, luteinizing hormone releasing hormone (LHRH) and [ d-Ala 6]-LHRH, and their metabolites in lung cell preparations. Isolated primary type II cells when grown in culture developed tight monolayers and exhibited morphologic characteristics of type I cells, as determined by transepithelial electrical resistance measurements and electron microscopy. The transformed type I-like cells exhibited a >10-fold decrease in proteolytic activities for LHRH, as compared to type II cells. The continuous lung cell line A549 formed leaky monolayers and exhibited similar enzyme activities to the primary type II cells. The responsible enzymes for degradation of LHRH in type II and A549 cells were angiotensin converting enzyme (ACE), EP24.11, and EP24.15. In contrast, no EP24.15 or ACE activity was observed in type I-like pneumocytes and only a weak EP24.11 activity was detected. In all cell types, the degradation rate of [ d-Ala 6]-LHRH was about 3–8 times lower than that of LHRH. This peptide analog was resistant to degradation by EP24.15 and EP24.11, but was susceptible to ACE-mediated cleavage.