title: Reverse transcription polymerase chain reaction (RT-PCR) analysis of proteolytic enzymes in cultures of human respiratory epithelial cells.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: School of Pharmacy and Pharmaceutical Sciences, Trinity College Dublin, Ireland.
authors.forename: Leonie
authors.initials: L
authors.lastname: Baginski
authors.forename: Gaëlle
authors.initials: G
authors.lastname: Tachon
authors.forename: Françoise
authors.initials: F
authors.lastname: Falson
authors.forename: John S
authors.initials: JS
authors.lastname: Patton
authors.forename: Udo
authors.initials: U
authors.lastname: Bakowsky
authors.forename: Carsten
authors.initials: C
authors.lastname: Ehrhardt
mesh_terms: D018938:Caco-2 Cells; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000201:enzymology*; D020869:Gene Expression Profiling / Q000379:methods*; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007413:Intestinal Mucosa / Q000201:enzymology; D010447:Peptide Hydrolases / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D020545:Respiratory Mucosa / Q000201:enzymology*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D012333:RNA, Messenger; D010447:Peptide Hydrolases