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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

Freeze-fracture replicas from TR-CSFB cells (A) and Z310 cells (B-D) treated with 1 μM dexamethsone (dexa. A, D) and 100 nM (B) and 550nM (C) hydrocortisone (HC). Whereas the TR-CSFB cells (A), but not the Z310 cells (D), have been partly induced by dexamethasone to form tight junctions (TJs), the Z310 cells (B,C), but not the TR-CSFB cells (not shown), can be induced by hydrocortisone to form some TJs. Arrows point to TJs particles in all pictures. EF: external fracture face, PF: protoplasmic fracture face.
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Figure 6: Freeze-fracture replicas from TR-CSFB cells (A) and Z310 cells (B-D) treated with 1 μM dexamethsone (dexa. A, D) and 100 nM (B) and 550nM (C) hydrocortisone (HC). Whereas the TR-CSFB cells (A), but not the Z310 cells (D), have been partly induced by dexamethasone to form tight junctions (TJs), the Z310 cells (B,C), but not the TR-CSFB cells (not shown), can be induced by hydrocortisone to form some TJs. Arrows point to TJs particles in all pictures. EF: external fracture face, PF: protoplasmic fracture face.

Mentions: Neither the untreated TR-CSFB nor Z310 cells showed any tight junctions in the freeze-fracture replica. In addition, the TR-CSFB cells did not react on hydrocortisone treatment with appearance of tight junctions (data not shown). However, dexamethasone treatment was answered by rare but consistent formation of tight junction networks associated predominantly with the P-face (fig. 6A). This result is consistent with the up-regulation of tight junction proteins in this cell line by dexamethasone (table 1, fig. 4). In contrast, and again in accordance with the biochemical data, dexamethasone did not increase the number of tight junctions in Z310 cells (fig. 6D), but in these cells tight junctions were slightly induced by hydrocortisone (fig. 6B, C). However, these tight junctions never formed continuous strands or networks, but insulated strands which probably would not increase the TEER. Indeed, this assumption was confirmed by the TEER values remaining constantly low after treatment with hydrocortisone or dexamethasone (fig. 5)


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)

Freeze-fracture replicas from TR-CSFB cells (A) and Z310 cells (B-D) treated with 1 μM dexamethsone (dexa. A, D) and 100 nM (B) and 550nM (C) hydrocortisone (HC). Whereas the TR-CSFB cells (A), but not the Z310 cells (D), have been partly induced by dexamethasone to form tight junctions (TJs), the Z310 cells (B,C), but not the TR-CSFB cells (not shown), can be induced by hydrocortisone to form some TJs. Arrows point to TJs particles in all pictures. EF: external fracture face, PF: protoplasmic fracture face.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Freeze-fracture replicas from TR-CSFB cells (A) and Z310 cells (B-D) treated with 1 μM dexamethsone (dexa. A, D) and 100 nM (B) and 550nM (C) hydrocortisone (HC). Whereas the TR-CSFB cells (A), but not the Z310 cells (D), have been partly induced by dexamethasone to form tight junctions (TJs), the Z310 cells (B,C), but not the TR-CSFB cells (not shown), can be induced by hydrocortisone to form some TJs. Arrows point to TJs particles in all pictures. EF: external fracture face, PF: protoplasmic fracture face.
Mentions: Neither the untreated TR-CSFB nor Z310 cells showed any tight junctions in the freeze-fracture replica. In addition, the TR-CSFB cells did not react on hydrocortisone treatment with appearance of tight junctions (data not shown). However, dexamethasone treatment was answered by rare but consistent formation of tight junction networks associated predominantly with the P-face (fig. 6A). This result is consistent with the up-regulation of tight junction proteins in this cell line by dexamethasone (table 1, fig. 4). In contrast, and again in accordance with the biochemical data, dexamethasone did not increase the number of tight junctions in Z310 cells (fig. 6D), but in these cells tight junctions were slightly induced by hydrocortisone (fig. 6B, C). However, these tight junctions never formed continuous strands or networks, but insulated strands which probably would not increase the TEER. Indeed, this assumption was confirmed by the TEER values remaining constantly low after treatment with hydrocortisone or dexamethasone (fig. 5)

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