Glial induction of blood-brain barrier-like L-system amino acid transport in the ECV304 cell line.
The blood-brain barrier (BBB) is formed by the presence of tight junction complexes between brain endothelial cells that restrict paracellular permeability. As a consequence, a number of transport proteins are expressed on cerebral endothelial cells to facilitate the transport of nutrients into the brain. Although the modulation of barrier tight junction properties by glial-conditioned medium and by second messengers is well established, little is known about the effects of these factors on carrier-mediated BBB transport processes. The ECV304 cell line shows an endothelial phenotype and can be induced to upregulate certain BBB features in the presence of glial factors. In the present study, we have examined the effect of conditioned medium derived from rat C6-glioma cells (C6CM) on the function of the L-system amino acid transporter in ECV304 cells, using L-leucine as the model substrate, and have determined whether the changes observed can be mimicked by modulating intracellular cAMP levels. ECV304 cells exposed to C6CM exhibited a significant increase in both the affinity of leucine transport and the diffusional constant (Michaelis-Menten), while the maximal transport capacity remained unchanged. Conversely, acute exposure to modulators of the PKA and PKC second messenger pathways was found to reduce significantly the maximal transport capacity and diffusion constants, while transport affinity remained unchanged. In both cases, the maximal flux of leucine was increased, indicating transport of greater efficiency. This study indicates that exposure of ECV304 cells to C6CM provides an influence inducing L-system transport properties characteristic of brain endothelial cells. Furthermore, it appears that L-system-mediated transport of amino acids can be modulated by several distinct pathways.
Blood-Brain Barrier Research Group, Centre for Neuroscience Research, GKT School of Biomedical Sciences, King's College, London, United Kingdom. mansor.chirsty@kcl.ac.uk
M
M
Chishty
A
A
Reichel
D J
DJ
Begley
N J
NJ
Abbott
D027062:Amino Acid Transport System L / Q000378:metabolism*; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001253:Astrocytes / Q000187:drug effects* / Q000378:metabolism; D001812:Blood-Brain Barrier / Q000187:drug effects* / Q000502:physiology; D001921:Brain / Q000187:drug effects* / Q000378:metabolism; D002772:Cholera Toxin / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D017077:Culture Media, Conditioned / Q000494:pharmacology; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D006133:Growth Substances / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D007930:Leucine / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D015290:Second Messenger Systems / Q000187:drug effects / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D027062:Amino Acid Transport System L; D000596:Amino Acids; D017077:Culture Media, Conditioned; D006133:Growth Substances; D005576:Colforsin; D002772:Cholera Toxin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D007930:Leucine; D013755:Tetradecanoylphorbol Acetate