Establishment of an in vitro brain barrier epithelial transport system for pharmacological and toxicological study.
An immortalized Z310 murine choroidal epithelial cell line was recently established in this laboratory. The purposes of this study were (1) to investigate the presence of tight junction (TJ) proteins in Z310 cells and (2) to develop a Z310 cell-based in vitro brain barrier transport model. Real-time RT-PCR studies revealed that Z310 cells possess mRNAs encoding ZO-1, -2, and -3, claudin-1, -2, -4, and -8, occludin, and connexin-32. Confocal microscopic analyses confirmed the presence of claudin-1 and ZO-1 in Z310 cells at cell-cell contact sites. When Z310 cells were grown on a two-chamber Transwell device, the [14C]sucrose permeability coefficient and transepithelial electrical resistance (TEER) across the cell monolayer were 6 x 10(-4) cm/min and 61 omega-cm2, respectively. To improve the tightness of Z310 barrier, the cells were cultured in astrocyte-conditioned medium (ACM), or in the presence of eicosapentaenoic acids (EPA, 10 microM), epidermal growth factor (EGF, 100 ng/mL), or dexamethasone (1 microM) in the growth medium. Treatment with ACM, EPA, EGF and dexamethasone significantly increased the TEER by 33%, 38%, 40%, and 50% above controls, respectively. However, only dexamethasone significantly reduced [14C]sucrose paracellular permeability (-231% of controls). These data suggest that Z310 cells possess the TJ proteins. The presence of dexamethasone in the growth medium improves the tightness of Z310 cell monolayer to the level better than that of the primary culture of choroidal epithelial cells. The Z310 cell-based in vitro model appears to be suitable for transepithelial transport study of drugs and toxicants.
Shi
Lewis Zhichang
LZ
School of Health Sciences, Purdue University, 550 Stadium Mall Drive, Room 1163D, West Lafayette, IN 47907-2051, USA.
Zheng
Wei
W
D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D001253:Astrocytes / Q000502:physiology; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology*; D001921:Brain / Q000166:cytology*; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D020099:Coated Materials, Biocompatible / Q000378:metabolism; D017630:Connexins / Q000378:metabolism*; D017077:Culture Media, Conditioned / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology; D004285:Dogs; D004815:Epidermal Growth Factor / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D005455:Fluorescent Antibody Technique / Q000379:methods; D005786:Gene Expression Regulation / Q000502:physiology; D006023:Glycoproteins / Q000494:pharmacology; D066298:In Vitro Techniques; D051379:Mice; D011228:Prealbumin; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013395:Sucrose / Q000493:pharmacokinetics; D019108:Tight Junctions / Q000187:drug effects / Q000378:metabolism*; D014316:Tritium / Q000493:pharmacokinetics
D000893:Anti-Inflammatory Agents; D020099:Coated Materials, Biocompatible; D017630:Connexins; D017077:Culture Media, Conditioned; D006023:Glycoproteins; D011228:Prealbumin; D012333:RNA, Messenger; C031458:embryonic prealbumin; D014316:Tritium; D013395:Sucrose; D004815:Epidermal Growth Factor; D003907:Dexamethasone