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Transport of [3H]MPP+ in an immortalized rat brain microvessel endothelial cell line (RBE 4)
F Martel1, C Calhau, P Soares-da-Silva
1Department of Biochemistry, Faculty of Medicine, Porto, Portugal. fatima_martel@hotmail.com
Abstract:
The aim of this study was to characterize the transport of the organic cation 1-methyl-4-phenylpyridinium (MPP+) in an immortalized cell line of rat capillary cerebral endothelial cells (RBE 4). Verapamil (100 microM) and rhodamine 123 (10 microM), and decynium22 (2 microM) and corticosterone (100 microM) reduced cellular accumulation of [3H]MPP+ applied from the luminal and abluminal cell border, respectively. When cells were grown on plastic supports, [3H]MPP+ accumulated in the cells. The kinetic parameters of the saturable component were: Km=25 microM and Vmax=246 pmol per mg protein and 15 min. A selective organic anion transport inhibitor and selective inhibitors of the L- and A-type amino acid transporters did not affect [3H]MPP+ uptake. Uptake of [3H]MPP+ was Na+-independent and metabolic energy-, pH- and potential-dependent. It was inhibited by several organic cations (e.g., verapamil, quinidine, daunomycin, dopamine) but not by others (cimetidine, tetraethylammonium, N-methylnicotinamide). In conclusion, [3H]MPP+ is efficiently transported by RBE 4 cells in both abluminal-to-luminal and luminal-to-abluminal directions. Absorption of [3H]MPP+ seems to occur through a carrier-mediated mechanism belonging to the amphiphilic solute facilitator (ASF) family of transporters, but distinct from the known members of this family.
Insights
This study investigated the transport of 1-methyl-4-phenylpyridinium (MPP+) in rat brain endothelial cells (RBE 4). Results indicate MPP+ uptake occurs via a carrier-mediated mechanism, likely a novel amphiphilic solute facilitator transporter.
Area of Science:
- Neuroscience
- Cell Biology
- Pharmacology
Background:
- The blood-brain barrier (BBB) tightly regulates the passage of substances into the brain.
- Understanding the transport mechanisms of organic cations across brain endothelial cells is crucial for drug delivery and understanding neurotoxicity.
Purpose of the Study:
- To characterize the transport of the organic cation 1-methyl-4-phenylpyridinium (MPP+) in an immortalized rat capillary cerebral endothelial cell line (RBE 4).
- To elucidate the kinetic parameters and identify potential transporters involved in MPP+ uptake and efflux.
Main Methods:
- Utilized [3H]MPP+ to study cellular accumulation in RBE 4 cells grown on plastic supports.
- Investigated the effects of various inhibitors (verapamil, rhodamine 123, decynium22, corticosterone, organic anion/amino acid transporter inhibitors, organic cations) on MPP+ uptake.
- Determined kinetic parameters (Km, Vmax) and assessed dependence on Na+, metabolic energy, pH, and membrane potential.
Main Results:
- [3H]MPP+ demonstrated saturable uptake with kinetic parameters Km=25 microM and Vmax=246 pmol/mg protein/15 min.
- Uptake was Na+-independent but dependent on metabolic energy, pH, and membrane potential.
- MPP+ transport was inhibited by organic cations like verapamil and quinidine, but not by others such as cimetidine.
- Transport occurred in both luminal-to-abluminal and abluminal-to-luminal directions.
Conclusions:
- RBE 4 cells efficiently transport [3H]MPP+ bidirectionally.
- MPP+ uptake is mediated by a carrier-driven mechanism, likely belonging to the amphiphilic solute facilitator (ASF) family.
- The identified transporter appears to be a novel member of the ASF family, distinct from currently known transporters.