Regulation of [(3)H]MPP(+) transport by phosphorylation/dephosphorylation pathways in RBE4 cells: role of ecto-alkaline phosphatase.
The aim of this study was to investigate the role of phosphorylation/dephosphorylation mechanisms at the blood-brain barrier (BBB) in the uptake of organic cations. The experiments were performed using RBE4 cells, an immortalized, rat brain microvessel endothelial cell line, an in vitro model of the BBB. The modulation of the uptake of 1-methyl-4-phenylpyridinium (MPP(+)), a model organic cation, at the apical membrane of RBE4 cells was studied. Agents that stimulate protein kinase A, but not protein kinase C, produced a moderate inhibition (approximately 18% reduction) of uptake of [(3)H]MPP(+) by RBE4 cells. Okadaic acid, an inhibitor of protein serine/threonine phosphatase, did not affect uptake of (3)H-MPP(+), but the alkaline phosphatase (ALP) inhibitor levamisole markedly reduced (3)H-MPP(+) uptake. The activity of membrane-bound ALP expressed on the apical surface of RBE4 cells was studied at pH 7.4 using p-nitrophenylphosphate as substrate. Kaempferol, progesterone, 3-isobutyl-1-methylxanthine, all- trans-retinoic acid and iron stimulated ecto-ALP activity and uptake of [(3)H]MPP(+) in RBE4. Orthovanadate (a protein tyrosine phosphatase inhibitor) markedly inhibited both ecto-ALP activity and uptake of [(3)H]MPP(+) by RBE4 cells. In conclusion, these results suggest that apical transporter(s) of MPP(+) in RBE4 cells may be under the control of phosphorylation/dephosphorylation mechanisms, being active in the dephosphorylated state. A physiological role for ALP in the modulation of organic cation transport in the BBB is suggested.
Calhau
C
C
Departamento de Bioquímica, Faculdade de Medicina. 4200-319 Porto, Portugal. ccalhau@med.up.pt
Martel
F
F
Soares-da-Silva
P
P
Hipólito-Reis
C
C
Azevedo
I
I
D015655:1-Methyl-4-phenylpyridinium / Q000378:metabolism / Q000493:pharmacokinetics*; D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D001812:Blood-Brain Barrier / Q000502:physiology*; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005110:Extracellular Space / Q000201:enzymology; D006868:Hydrolysis; D007700:Kinetics; D007978:Levamisole / Q000494:pharmacology; D019319:Okadaic Acid / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D014638:Vanadates / Q000494:pharmacology
D004791:Enzyme Inhibitors; D019319:Okadaic Acid; D007978:Levamisole; D014638:Vanadates; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D000469:Alkaline Phosphatase; D010749:Phosphoprotein Phosphatases; D015655:1-Methyl-4-phenylpyridinium