Specific aminopeptidases of excised human nasal epithelium and primary culture: a comparison of functional characteristics and gene transcripts expression.
OBJECTIVE
To investigate whether growing human nasal epithelium as primary cultures alters aminopeptidase B (APB), aminopeptidase N (APN) and dipeptidyldipeptidase (DPPIV) metabolic characteristics, and mRNA gene transcript expression.


METHODS
The formation of 7-amino-methyl coumarin from specific substrates for APN (L-alanine-4-methyl-coumaryl-7-amide, APB (L-arginine-4-methyl-coumaryl-7-amide) and DPPIV (glycyl-L-proline-4-methyl-coumaryl-7-amide) was used to estimate the KM, Vmax and the effect of aminopeptidases inhibitors on the enzymes. Polymerase chain reaction was used to investigate gene expression.


RESULTS
Results of this study showed that: (1) both the excised tissues and primary cultures of human nasal epithelium expressed APN, APB and DPPIV activity; (2) the KM of APB, APN and DPPIV was not significantly different in cell and tissue homogenates; (3) except for APN, the Vmax was not significantly different in the two metabolism models; (4) there was no statistically significant difference in the behaviours of APB, APN and DPPIV in response to inhibition by puromycin and bestatin in the two models; (5) the mRNA transcripts that encode APB, APN and DPPIV were expressed in both cell culture and tissue homogenate.


CONCLUSIONS
Based on the results of this study, it may be concluded that nasal primary culture system is suitable for investigating peptide and protein metabolism and enzymatic stability in human nasal epithelium. Except for APN, the tissue culture conditions did not significantly alter the functional and molecular expression of the aminopeptidases.
Agu
Remigius U
RU
Biopharmaceutics and Drug Delivery Laboratory, College of Pharmacy, Department for Human Genetics, Katholieke Universiteit Leuven, Leuven, Belgium. remigius.agu@dal.ca
Obimah
Dominic U
DU
Lyzenga
Wendy J
WJ
Jorissen
Mark
M
Massoud
Emad
E
Verbeke
Norbert
N
D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D018826:CD13 Antigens / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D003374:Coumarins / Q000378:metabolism; D018819:Dipeptidyl Peptidase 4 / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000201:enzymology; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D009297:Nasal Mucosa / Q000166:cytology / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D011480:Protease Inhibitors / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011691:Puromycin / Q000494:pharmacology
C502573:4-methyl-coumaryl-7-amide; D003374:Coumarins; D011480:Protease Inhibitors; D011500:Protein Synthesis Inhibitors; D011691:Puromycin; D000626:Aminopeptidases; D018826:CD13 Antigens; C019721:aminopeptidase B; D018819:Dipeptidyl Peptidase 4; D007930:Leucine; C012211:ubenimex