Reverse Transcription Polymerase Chain Reaction (RT-PCR) Analysis of Proteolytic Enzymes in Cultures of Human Respiratory Epithelial Cells

• Published in: Journal of aerosol medicine Vol. 24; no. 2; pp. 89 - 101
• Main Authors: BAGINSKI, Leonie, TACHON, Gaëlle, FALSON, Françoise, PATTON, John S, BAKOWSKY, Udo, EHRHARDT, Carsten
• Format: Journal Article
• Language: English
• Published: New Rochelle, NY Mary Ann Liebert 01.04.2011; Mary Ann Liebert, Inc
• Subjects: Aerosols; Alveoli; Aminopeptidase; Aspartic proteinases; Biological and medical sciences; Caco-2 Cells; Carboxypeptidase; Cell culture; Cells, Cultured; Digestive enzymes; Drug delivery; Elastase; endopeptidase; Enteropeptidase; Epithelial cells; Epithelial Cells - enzymology; gamma -Glutamyltransferase; Gene expression; Gene Expression Profiling - methods; Gene Expression Regulation, Enzymologic; General pharmacology; Genetic aspects; Health technology assessment; Humans; Intestinal Mucosa - enzymology; Leukocytes (neutrophilic); Lung; Medical sciences; Meprin A; Neprilysin; Pancreas; peptidase; Peptide Hydrolases - genetics; Pharmaceutical technology. Pharmaceutical industry; Pharmaceuticals; Pharmacology. Drug treatments; Physiological aspects; Polymerase chain reaction; Practice; Proteinase; Proteolysis; Proteolytic enzymes; Registered nurses; Respiration; Respiratory Mucosa - enzymology; Reverse Transcriptase Polymerase Chain Reaction; Reverse transcription; RNA, Messenger - metabolism; Spatial distribution; Thimet oligopeptidase; Trypsin; France; Germany; Human; Pharmaceutical technology; Peptides; Enzyme; Lung; A549; proteases; pulmonary peptide and protein delivery; Reverse transcription; Protein; 16HBE14o; human lung epithelial cells; mRNA expression; Polymerase chain reaction; Peptidases; proteolytic enzymes; Messenger RNA; Calu-3; Caco-2; Hydrolases; Epithelial cell
• ISSN: 1941-2711; 1941-2703; 1941-2703
• DOI: 10.1089/jamp.2010.0842

Pancreatic proteolytic digestive enzymes are a major extracellular barrier to the sucessful systemic delivery of biopharmaceuticals via the oral route, whereas in health in the lungs these powerful proteases are virtually absent from the extracellular fluids. Despite this, the absorption of some (but not all) natural peptides and proteins from the lungs may be poor, and one has to acknowledge that information on the activity and spatial distribution of proteolytic enzymes in the human lung is scarce. Here, we investigated expression patterns of a series of proteolytic enzymes in several human respiratory cell types on mRNA level in an attempt to better understand the fate of inhaled biopharmaceuticals. The mRNA expression of proteolytic enzymes (i.e., carboxypeptidases: CPA1, CPA2, CPB, CPM; gamma-glutamyltransferases: GGT1, GGT2; angiotensin-converting enzymes: ACE, ACE2; aminopeptidases: APA, APB, APN, APP1, APP2, APP3; endopeptidases: 24.11 (neprilysin), 24.15 (thimet oligopeptidase), 24.18 (meprin A); enteropeptidase; trypsin 1, trypsin 2; neutrophilic elastase; dipeptidyl peptidase 4; gamma-glutamylhydrolase) was investigated by semiquantitative RT-PCR in human bronchial (hBEpC, Calu-3, 16HBE14o-) and alveolar (A549) epithelial cells, respectively. Gastrointestinal Caco-2 cells were used as comparison. Obvious differences were observed in proteinases' expression pattern between the investigated cell types. Although considered to be of bronchial epithelial phenotype, neither Calu-3 nor 16HBE14o- cells matched the mRNA expression pattern of hBEpC in primary culture. Of all investigated cell lines, Caco-2 expresses the highest number of proteases and peptidases. Although mRNA expression does not necessarily signify enzyme functionality, our results provide the first comprehensive analysis of peptidase and protease expression and distribution in human lung epithelial cells and are the basis for further investigations.