Characterisation of the R3/1 cell line as an alveolar epithelial cell model for drug disposition studies.
The rat cell line R3/1 displays several phenotypical features of alveolar epithelial type I cells. In order to evaluate this cell line as potential in vitro model for drug disposition studies, R3/1 cells were cultured on Transwell filters and the transepithelial electrical resistance (TEER) was measured to test the integrity of cell layers. The mRNA expression of cell junctional components including E-cadherin, occludin, ZO-1 and ZO-2 was studied using reverse transcriptase-polymerase chain reaction (RT-PCR) and the corresponding proteins by immunofluorescence microscopy (IFM). Moreover, the expression pattern of catabolic peptidases, carboxypeptidase M, aminopeptidases (AP): A, B, N and P, gamma-glutamyltransferase (GGT), dipeptidylpeptidase IV, angiotensin-converting enzyme (ACE), and endopeptidases (EP) 24.11 and 24.15 was analysed in R3/1 cells and compared to rat alveolar epithelial I-like cells in primary culture. TEER peaked at 99+/-17Omegacm(2) after 5 days in culture. Addition of 0.1muM dexamethasone (DEX) with 20% foetal bovine serum further increased TEER by 65%. However, none of the culture conditions used in our study yielded monolayers with TEER values comparable to those of primary cultures of rat pneumocytes. No transcripts encoding for E-cadherin and occludin were detected by RT-PCR. However, ZO-1 and -2 mRNA transcripts were found. IFM using a monoclonal antibody against occludin confirmed the absence of the protein in R3/1 cells. Of the investigated proteolytic enzymes, mRNA transcripts encoding APA and APB as well as EP 24.11 and EP 24.15 were detected; a pattern similar to that of rat alveolar epithelial I-like cells in primary culture. Thus, although R3/1 cells express certain markers typical for type I pneumocytes (e.g., T1alpha, ICAM-1, connexin-43, caveolins-1 and -2) they do not form electrically tight monolayers. This excludes R3/1 cells from being used as an in vitro model for alveolar absorption. However, the cell line may be suitable to study stability of inhaled and endogenous proteins.
Horálková
Lenka
L
Trinity College Dublin, School of Pharmacy and Pharmaceutical Sciences, Westland Row, Dublin 2, Ireland.
Radziwon
Aneta
A
Endter
Sibylle
S
Andersen
Rikke
R
Koslowski
Roland
R
Radomski
Marek W
MW
Dolezal
Pavel
P
Ehrhardt
Carsten
C
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D017931:DNA Primers; D004587:Electrophoresis, Agar Gel; D008297:Male; D008565:Membrane Proteins / Q000235:genetics; D008856:Microscopy, Fluorescence; D010599:Pharmacokinetics; D010750:Phosphoproteins / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein
D017931:DNA Primers; D008565:Membrane Proteins; D010750:Phosphoproteins; D012333:RNA, Messenger; C569044:Tjp1 protein, rat; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein