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Modulation of multidrug resistance protein expression in porcine brain capillary endothelial cells in vitro
Abstract:
Multidrug resistance-associated protein (MRP) is a transport system that is involved in the elimination of xenobiotics and biologically active endogenous substrates. Recently, the presence of MRP has been demonstrated in cultured brain capillary endothelial cells (BCECs). The time-dependent, functional expression of MRP in porcine BCECs was investigated to assess the value of this cell culture model for drug transport at the blood-brain barrier. Western blot analysis was used to investigate MRP expression in freshly isolated porcine BCECs and compared to MRP expression at days 8 and 10 in culture. Subcellular localization of MRP was investigated by immunocytochemistry with an MRP-specific monoclonal antibody, MRPr1. Functional activity of MRP was assessed by efflux studies with the fluorescent MRP substrate glutathione-methylfluorescein (GS-MF). No significant MRP expression was detected in freshly isolated endothelial cells. However, MRP expression is up-regulated in cell culture in a time-dependent manner. Immunostaining revealed predominantly perinuclear and, to a lesser degree, plasma membrane localization of MRP. At 10 degrees C GS-MF efflux was significantly decreased, indicating the involvement of an energy-dependent transport system. Efflux of GS-MF was apparently inhibited by MK571, a specific inhibitor for MRP. Porcine BCECs demonstrate up-regulation of functional MRP expression during culture, as observed in human tissue, and therefore might serve as a useful in vitro system for studying MRP-mediated blood-brain barrier transport.
Insights
Multidrug resistance-associated protein (MRP) expression increases over time in cultured porcine brain capillary endothelial cells (BCECs). This functional upregulation suggests BCECs are a valuable model for studying blood-brain barrier drug transport.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- Multidrug resistance-associated protein (MRP) facilitates xenobiotic and endogenous substrate elimination.
- MRP presence has been confirmed in brain capillary endothelial cells (BCECs).
Purpose of the Study:
- To investigate the time-dependent, functional expression of MRP in porcine BCECs.
- To evaluate porcine BCECs as an in vitro model for blood-brain barrier (BBB) drug transport.
Main Methods:
- Western blot analysis for MRP expression in freshly isolated and cultured porcine BCECs.
- Immunocytochemistry using an MRP-specific antibody (MRPr1) for subcellular localization.
- Efflux studies with glutathione-methylfluorescein (GS-MF) to assess MRP functional activity.
Main Results:
- No significant MRP expression was detected in freshly isolated BCECs.
- MRP expression significantly increased in BCECs cultured over time (days 8 and 10).
- Immunostaining showed MRP localized perinuclearly and at the plasma membrane.
- GS-MF efflux decreased at 10°C, indicating energy-dependent transport.
- MK571, an MRP inhibitor, reduced GS-MF efflux, confirming MRP's role.
Conclusions:
- Porcine BCECs exhibit time-dependent upregulation of functional MRP expression in culture.
- This upregulation mirrors findings in human tissue.
- Porcine BCECs represent a promising in vitro model for investigating MRP-mediated transport at the blood-brain barrier.
