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Characterization of immortalized choroid plexus epithelial cell lines for studies of transport processes across the blood-cerebrospinal fluid barrier.

Kläs J, Wolburg H, Terasaki T, Fricker G, Reichel V - Cerebrospinal Fluid Res (2010)

Bottom Line: Primary cells and cell lines showed similar, but overall lower expression of Abc transporters and absent Slc expression when compared to intact tissue.Consistent with this Mrp1, Mrp4 and P-gp protein levels were higher in intact CP compared to cell lines.Taken together, immortalized cell lines may be a tool to study transport processes mediated by P-gp, Mrp1 or Mrp4, but overall expression of transport proteins and TJ formation do not reflect the situation in intact CP tissue.

View Article: PubMed Central - HTML - PubMed

Affiliation: Ruprecht-Karls University, Department of Pharmaceutical Technology, 69120 Heidelberg, Germany. reichel@uni-hd.de.

ABSTRACT

Background: Two rodent choroid plexus (CP) epithelial cell lines, Z310 and TR-CSFB, were compared with primary rat CP epithelial cells and intact CP tissue with respect to transport protein expression, function and tight junction (TJ) formation.

Methods: For expression profiles of transporters and TJ proteins, qPCR and western blot analysis were used. Uptake assays were performed to study the functional activity of transporters and TJ formation was measured by trans-epithelial electrical resistance (TEER) and visualized by electron microscopy.

Results: The expression of known ATP-binding cassette (Abc) transporter and solute carrier (Slc) genes in CP was confirmed by qPCR. Primary cells and cell lines showed similar, but overall lower expression of Abc transporters and absent Slc expression when compared to intact tissue. Consistent with this Mrp1, Mrp4 and P-gp protein levels were higher in intact CP compared to cell lines. Functionality of P-gp and Mrp1 was confirmed by Calcein-AM and CMFDA uptake assays and studies using [3H]bis-POM-PMEA as a substrate indicated Mrp4 function. Cell lines showed low or absent TJ protein expression. After treatment of cell lines with corticosteroids, RNA expression of claudin1, 2 and 11 and occludin was elevated, as well as claudin1 and occludin protein expression. TJ formation was further investigated by freeze-fracture electron microscopy and only rarely observed. Increases in TJ particles with steroid treatment were not accompanied by an increase in transepithelial electrical resistance (TEER).

Conclusion: Taken together, immortalized cell lines may be a tool to study transport processes mediated by P-gp, Mrp1 or Mrp4, but overall expression of transport proteins and TJ formation do not reflect the situation in intact CP tissue.

No MeSH data available.


Related in: MedlinePlus

Western blot analyses indicate expression of Mrp1, 4 and P-gp. Whole protein was used from freshly isolated CP and membrane vesicles were isolated from cells (20 μg). There are slight differences in protein size possibly due to modifications in tissue and cells during processing.
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Figure 2: Western blot analyses indicate expression of Mrp1, 4 and P-gp. Whole protein was used from freshly isolated CP and membrane vesicles were isolated from cells (20 μg). There are slight differences in protein size possibly due to modifications in tissue and cells during processing.

Mentions: Membrane proteins were isolated from the cell lines, while whole protein was used from rat CP, because, due to its small size, membrane isolation was not possible. CP tissue functioned as a positive control, Protein expression was not visualized in primary cells because we wanted to concentrate on characterization of the two immortalized cell lines. Abc transporters which showed expression at the RNA level in the cell lines were investigated for proteins expression. Mrp1, 4 and P-gp proteins were found in all three samples (fig. 2), and therefore further investigated for functional activity. Mrp5 could not be detected in the cell lines, only in freshly isolated CP (data not shown), therefore we did not consider Mrp5 to be relevant in the uptake studies of Mrp4. Mrp2 that was expressed at RNA level in TR-CSFB cells could not be detected at the protein level (data not shown).


Characterization of immortalized choroid plexus epithelial cell lines for studies of transport processes across the blood-cerebrospinal fluid barrier.

Kläs J, Wolburg H, Terasaki T, Fricker G, Reichel V - Cerebrospinal Fluid Res (2010)

Western blot analyses indicate expression of Mrp1, 4 and P-gp. Whole protein was used from freshly isolated CP and membrane vesicles were isolated from cells (20 μg). There are slight differences in protein size possibly due to modifications in tissue and cells during processing.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2927495&req=5

Figure 2: Western blot analyses indicate expression of Mrp1, 4 and P-gp. Whole protein was used from freshly isolated CP and membrane vesicles were isolated from cells (20 μg). There are slight differences in protein size possibly due to modifications in tissue and cells during processing.
Mentions: Membrane proteins were isolated from the cell lines, while whole protein was used from rat CP, because, due to its small size, membrane isolation was not possible. CP tissue functioned as a positive control, Protein expression was not visualized in primary cells because we wanted to concentrate on characterization of the two immortalized cell lines. Abc transporters which showed expression at the RNA level in the cell lines were investigated for proteins expression. Mrp1, 4 and P-gp proteins were found in all three samples (fig. 2), and therefore further investigated for functional activity. Mrp5 could not be detected in the cell lines, only in freshly isolated CP (data not shown), therefore we did not consider Mrp5 to be relevant in the uptake studies of Mrp4. Mrp2 that was expressed at RNA level in TR-CSFB cells could not be detected at the protein level (data not shown).

Bottom Line: Primary cells and cell lines showed similar, but overall lower expression of Abc transporters and absent Slc expression when compared to intact tissue.Consistent with this Mrp1, Mrp4 and P-gp protein levels were higher in intact CP compared to cell lines.Taken together, immortalized cell lines may be a tool to study transport processes mediated by P-gp, Mrp1 or Mrp4, but overall expression of transport proteins and TJ formation do not reflect the situation in intact CP tissue.

View Article: PubMed Central - HTML - PubMed

Affiliation: Ruprecht-Karls University, Department of Pharmaceutical Technology, 69120 Heidelberg, Germany. reichel@uni-hd.de.

ABSTRACT

Background: Two rodent choroid plexus (CP) epithelial cell lines, Z310 and TR-CSFB, were compared with primary rat CP epithelial cells and intact CP tissue with respect to transport protein expression, function and tight junction (TJ) formation.

Methods: For expression profiles of transporters and TJ proteins, qPCR and western blot analysis were used. Uptake assays were performed to study the functional activity of transporters and TJ formation was measured by trans-epithelial electrical resistance (TEER) and visualized by electron microscopy.

Results: The expression of known ATP-binding cassette (Abc) transporter and solute carrier (Slc) genes in CP was confirmed by qPCR. Primary cells and cell lines showed similar, but overall lower expression of Abc transporters and absent Slc expression when compared to intact tissue. Consistent with this Mrp1, Mrp4 and P-gp protein levels were higher in intact CP compared to cell lines. Functionality of P-gp and Mrp1 was confirmed by Calcein-AM and CMFDA uptake assays and studies using [3H]bis-POM-PMEA as a substrate indicated Mrp4 function. Cell lines showed low or absent TJ protein expression. After treatment of cell lines with corticosteroids, RNA expression of claudin1, 2 and 11 and occludin was elevated, as well as claudin1 and occludin protein expression. TJ formation was further investigated by freeze-fracture electron microscopy and only rarely observed. Increases in TJ particles with steroid treatment were not accompanied by an increase in transepithelial electrical resistance (TEER).

Conclusion: Taken together, immortalized cell lines may be a tool to study transport processes mediated by P-gp, Mrp1 or Mrp4, but overall expression of transport proteins and TJ formation do not reflect the situation in intact CP tissue.

No MeSH data available.


Related in: MedlinePlus